Devices and methods for separating circulating tumor cells from biological samples

ABSTRACT

A variety of devices, systems, kits, and methods are provided for separating or enriching circulating tumor cells in a biological sample such as whole blood. In some aspects, the devices are multi-stage devices including at least a filtering stage, and two separation stages for ferrohydrodynamic separation of magnetically labelled white blood cells and for marker-independent and size-independent focusing of magnetically labeled particles so as to separate or enrich unlabeled rare cells in the biological sample. The devices and methods are, in some aspects, capable of high throughput in excess of 6 milliliters per hour while achieving high separation (&gt;97%) of the unlabeled rare cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.provisional application entitled “MICROFLUIDIC MAGNETIC CELL SORTER FORBREAST CANCER CIRCULATING TUMOR CELLS” having Ser. No. 62/668,355, filedMay 8, 2018 and U.S. provisional application entitled “DEVICES ANDMETHODS FOR SEPARATING CIRCULATING TUMOR CELLS FROM BIOLOGICAL SAMPLES”having Ser. No. 62/826,539, filed Mar. 29, 2019, both of which areincorporated by reference in their entirety. This application is also acontinuation-in-part of and claims benefit of co-pending PCT applicationentitled “DEVICES AND METHODS FOR SEPARATING CIRCULATING TUMOR CELLSFROM BIOLOGICAL SAMPLES” having International Application No.PCT/US18/45294, filed Aug. 4, 2018, which claims priority to U.S.provisional application entitled “CELL SEPARATION OF TUMOR CELLS” havingSer. No. 62/541,552, filed Aug. 4, 2017, all of which are incorporatedby reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under award 1150042 andaward 1359095 and 1659525 awarded by the National Science Foundation andaward R21GM104528 awarded by the National Institutes of Health and awardUL1TR002378 awarded by the National Center for Advancing TranslationalSciences of the National Institutes of Health. The government hascertain rights in the invention.

TECHNICAL FIELD

The present disclosure generally relates to microfluidics and usesthereof.

BACKGROUND

Circulating tumor cells (CTCs) are cancer cells that are detached fromprimary solid tumors and carried through the vasculature to potentiallyseed distant site metastases in vital organs, representing the maincause of death in cancer patients. Molecular assessments of CTCs notonly could benefit basic cancer research, but also might eventually leadto a more effective cancer treatment. However, one major limitation hasbeen the limited availability of viable CTCs for investigations, due inpart to the small patient blood volumes that are allowable for research,which usually yielded less than 100 CTCs from 1 mL of whole blood. As aresult, technologies are needed in order to separate these rare cellsfrom blood, and important performance criteria for these technologiesinclude the ability to process a significant amount of blood quickly(e.g., throughput ˜7.5 mL h⁻¹), a high recovery rate of CTCs, areasonable purity of isolated cancer cells, and cell integrity forfurther characterization.

Label-based CTC separation technologies were developed to selectivelyenrich a subset of CTCs from blood, primarily through the use ofspecific biological markers including epithelial cell adhesion molecule(EpCAM) (FIG. 1A). These antigen-based labels were a rate-limitingfactor in effective CTC separation, as the inherent heterogeneity ofCTCs render these technologies ineffective for general use. The vastarray of various biomarkers that might or might not be expressed, andwhich cannot be predicted to remain expressed in CTCs undergoingEpithelial-to-Mesenchymal Transitions (EMT) are cumbersome andconfounding in these label-based methods. Furthermore, most label-basedtechnologies do not conveniently enable comprehensive molecular analysisof separated CTCs because they are either dead or immobilized to asurface. Thus, a variety of label-free methods have been developed toexploit specific physical markers in order to deplete non-CTCs in bloodand thereby enrich cancer cells. While such methods may be used toseparate CTCs based upon, for example, size, the existence of largewhite blood cells, such as monocytes, that may have overlapping sizeswith CTCs complicate these label-free methods and reduce the purity ofthe sample obtained. Other devices have attempted to incorporate two ormore of these methods, but still suffer from the time-consuming andlaborious sample preparation due to the complications discussed abovefor labeling CTCs.

There remains a need for improved devices and methods for separatingcirculating tumor cells that overcome the aforementioned deficiencies.

SUMMARY

In various aspects, microfluidic devices and methods of usingmicrofluidic devices are provided that overcome one or more of theaforementioned deficiencies. The devices and methods can combinefilters, sheathing separation, and flow focusing to provide for highthroughput cell separation without the need for labeling the CTCs.

The present disclosure provides cell separation system and devices forenriching circulating tumor cells in a biological sample. In aspects,the system includes a plurality of magnetic microbeads for conjugationto white blood cells in the sample, a biocompatible superparamagneticsheathing composition, and a multi-stage microfluidic device comprisingfirst, second, and third microfluidic channels, and comprising one ormore magnetic sources, the device configured to remove waste particlesfrom the sample, remove a plurality of magnetically conjugated whiteblood cells from the sample, and to enrich circulating tumor cells inthe sample. In embodiments of the systems of the present disclosure,included are a plurality of magnetic microbeads adapted for conjugationto white blood cells in the biological sample and not for conjugationwith the circulating tumor cells configured to provide a magneticallylabeled biological sample having a majority of the white blood cells inthe biological sample conjugated to one or more magnetic microbeads. Inaspects of systems of the present disclosure, the biocompatiblesuperparamagnetic sheathing composition comprises a plurality ofmagnetic nanoparticles and a biocompatible surfactant, the biocompatiblesuperparamagnetic sheathing composition adapted to be combined with abiocompatible carrier fluid to make a biocompatible superparamagneticsheathing fluid.

In various aspects, the multi-stage microfluidic device of systems ofthe present disclosure includes: (i) a filter section comprising a firstmicrofluidic channel, a first fluid inlet, and one or more filters alonga length of the first microfluidic channel, wherein the first fluidinlet is configured to receive the magnetically labeled biologicalsample, and wherein the one or more filters are configured to remove afirst plurality of waste particles from the magnetically labeledbiological sample; (ii) a first separation stage comprising a secondmicrofluidic channel fluidly connected to the first microfluidic channeland having a second fluid inlet to receive the biocompatiblesuperparamagnetic sheathing fluid, and a first fluid outlet at an end ofthe second microfluidic channel and offset from a central diameter ofthe second microfluidic channel, wherein the second microfluidic channelis configured to combine the magnetically labeled biological sample fromthe first filter section with the biocompatible superparamagneticsheathing fluid; (iii) a second separation stage comprising a thirdmicrofluidic channel fluidly connected to the second microfluidicchannel and having a second fluid outlet and at least a firstcirculating tumor cell outlet and second circulating tumor cell outlet;and (iv) one or more magnetic sources adjacent to the multi-stagemicrofluidic device. In aspects, the one or more magnetic sources areconfigured to produce: (a) a non-uniform magnetic field along the secondmicrofluidic channel having a component sufficiently perpendicular to alength of the second microfluidic channel to cause a plurality of whiteblood cells conjugated to the magnetic beads to be deflected into thefirst fluid outlet; and (b) a substantially symmetric magnetic fieldhaving a field maximum along a length of the third microfluidic channelsufficient to cause the white blood cells conjugated to the magneticbeads in the third microfluidic channel to be focused toward a center ofthe third microfluidic channel and to exit the channel via the secondfluid outlet and to cause circulating tumor cells to be deflectedtowards an outer portion of the third microfluidic channel and to exitthe channel via the first or second circulating tumor cell outlet.

The present disclosure also provides multi-stage microfluidic devicesfor enriching circulating tumor cells in a biological sample. In variousembodiments, the devices of the present disclosure include: (i) a filtersection comprising a first microfluidic channel, a first fluid inlet,and one or more filters along a length of the first microfluidicchannel, wherein the first fluid inlet is configured to receive abiological sample comprising circulating tumor cells and white bloodcells, wherein a majority of the white blood cells in the biologicalsample are conjugated to magnetic microbeads, and wherein the one ormore filters are configured to remove a first plurality of wasteparticles from the biological sample; (ii) a first separation stagecomprising a second microfluidic channel fluidly connected to the firstmicrofluidic channel and having a second fluid inlet to receive abiocompatible superparamagnetic sheathing fluid, and a first fluidoutlet at an end of the second microfluidic channel opposite the secondfluid inlet and offset from a central diameter of the secondmicrofluidic channel, wherein the second microfluidic channel isconfigured to combine the biological sample from the first filtersection with the biocompatible superparamagnetic sheathing fluid, andwherein the biocompatible superparamagnetic sheathing fluid comprises aplurality of magnetic nanoparticles and a biocompatible surfactantsuspended in a biocompatible carrier fluid; (iii) a second separationstage comprising a third microfluidic channel fluidly connected to thesecond microfluidic channel and having a second fluid outlet and atleast a first circulating tumor cell outlet and second circulating tumorcell outlet; and (iv) one or more magnetic sources adjacent to themulti-stage microfluidic device and configured to produce: (a) anon-uniform magnetic field along the second microfluidic channel havinga component sufficiently perpendicular to a length of the secondmicrofluidic channel to cause a plurality of white blood cellsconjugated to the magnetic beads to be deflected into the first fluidoutlet; and (b) a substantially symmetric magnetic field having a fieldmaximum along a length of the third microfluidic channel sufficient tocause the white blood cells conjugated to the magnetic beads in thethird microfluidic channel to be focused toward a center of the thirdmicrofluidic channel and to exit the channel via the second fluid outletand to cause circulating tumor cells to be deflected towards an outerportion of the third microfluidic channel and to exit the channel viathe first or second circulating tumor cell outlet.

Methods of enriching circulating tumor cells in a biological samplecomprising a plurality of components are also provided in the presentdisclosure. According to various aspects, the method comprises:combining the biological sample with a plurality of magnetic microbeadsadapted to conjugate to white bloods cells such that a majority of whiteblood cells in the sample become conjugated to at least one magneticmicrobead to produce a magnetically labeled biological sample;introducing the magnetically labeled biological sample into the firstfluid inlet of a microfluidic device according the present disclosure;introducing the biocompatible superparamagnetic sheathing fluid into thesecond fluid inlet of the microfluidic device, such that themagnetically labeled biological sample combines with the biocompatiblesuperparamagnetic sheathing fluid to produce a combined fluid sample;and causing the combined fluid sample to flow along the firstmicrofluidic channel, the second microfluidic channel, and the thirdmicrofluidic channel such that a majority of the magnetic microbeads andwhite blood cells are deflected to the first or second fluid outlet anda majority of the circulating tumor cells from the biological sample arecollected in the one or more circulating tumor cell outlets.

The present disclosure also provides methods of enriching circulatingtumor cells in a sample of whole blood, where the whole blood comprisesunlabeled rare cells and white blood cells. In various aspects, themethod includes: (i) adding a plurality of magnetic beads to the sampleto produce a magnetically labeled sample, wherein a majority of thewhite blood cells are associated with the magnetic beads and wherein themagnetic beads do not conjugate to the unlabeled rare cells; (ii)filtering the magnetically labeled sample in a filter section of amicrofluidic device to produce a filtered sample by removing largedebris from the magnetically labeled sample; (iii) separating at least aportion of the white blood cells that are associated with the magneticbeads by flowing the filtered sample in a first separation stage of themicrofluidic device through a sheath flow in a biocompatiblesuperparamagnetic sheathing fluid in a non-uniform magnetic field toproduce a first enriched sample; and (iv) isolating a majority of theunlabeled rare cells by flowing the first enriched sample through achannel in a second separation stage of the microfluidic device with asubstantially symmetric focused magnetic field such that a majority ofany remaining white blood cells associated with the magnetic beads arefocused towards a center of the channel and to a central channel outletand the majority of the unlabeled rare cells are collected from one ormore peripheral channel outlets.

Other systems, methods, devices, features, and advantages of the devicesand methods will be or become apparent to one with skill in the art uponexamination of the following drawings and detailed description. It isintended that all such additional systems, methods, devices, features,and advantages be included within this description, be within the scopeof the present disclosure, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1.1A is a schematic illustration of traditional label-basedmagnetophoresis for CTC separation, in which rare cells were targetedvia specific biomarkers such as epithelial cell adhesion molecule(EpCAM) through functionalized magnetic particles in order to pull thesecells through magnetic force towards magnetic field maxima in acontinuous-flow manner. FIG. 1.1B is a schematic illustration oflabel-free ferrohydrodynamic cell separation (FCS) for CTCs. Large rarecells in ferrofluid experience large magnetic buoyance force and will bepushed towards magnetic field minimum FIG. 1.1C is a schematic ofintegrated ferrohydrodynamic cell separation (iFCS) for CTC separation.In iFCS, RBC-lysed blood and biocompatible ferrofluid were processed ina single straight channel. CTCs of different size and labelled WBCs werepushed toward two different directions, resulting in a spatialseparation at the end of the iFCS device. FIG. 1.1D is a photograph of aprototype iFCS device filled with dye and a permanent magnet. FIG. 1.1Eis a top-view of the iFCS device with labels of inlets, outlets. FIG.1.1F is a simulation of magnetic flux density and particle trajectoriesof magnetic particles and non-magnetic particles in channel (L×W×H,57.80 mm×0.90 mm×0.15 mm)

FIGS. 1.2A-1.2I show optimization of iFCS device via simulation andbeads calibration. A 3D model was developed to simulate particletrajectories and validated by comparing simulation and experimentalresults. Numerical optimization of final position Y, separation distanceΔY, and the expected range (standard deviation) at the end of thechannel was conducted with different parameters: (FIGS. 1.2A, 1.2D, and1.2G) throughput, (FIGS. 1.2B, 1.2E, and 1.2H) ferrofluid concentration,and (FIGS. 1.2C, 1.2F, and 1.2I) magnetic field gradient. Ferrofluidconcentration was fixed at 0.292% for FIG. 1.2A, FIG. 1.2D, FIG. 1.2G,FIG. 1.2C, FIG. 1,2F, and FIG. 1.2I. Magnetic field gradient was fixedat 132 T m⁻¹ for FIGS. 1.2A, 1.2D, and 1.2G. Throughput was fixed at 100μL min⁻¹ for FIGS. 1.2B, 1.2E, 1.2, 1.2C, 1.2F, and 1.2I. FIGS. 1.2D,1.2E, and 1.2F were simulated particles positions and correspondingstandard deviations. FIGS. 1.2G, 1.2H, and 1.2I were normalized particledistributions in experiment.

FIGS. 1.3A-1.3F demonstrate iFCS applied to multiple beads separation.FIG. 1.3A shows average separation distance between non-magnetic beadsand magnetic beads. Non-magnetic beads (20.3 μm, 8.0 μm and 5.7 μm) andmagnetic beads (11.8 μm) were spiked into 0.292% ferrofluid atthroughput between 10 and 350 μL min⁻¹. The magnetic field gradient wasfixed at 132 T m⁻¹. FIG. 1.3B illustrates simulated and experimentalseparation distance between 8.0 μm non-magnetic beads and 11.8 μmmagnetic beads at low ferrofluid concentration between 0.01% and 0.1%,and flow rate between 10 and 210 μL min⁻¹. The magnetic field gradientwas fixed at 132 T m⁻¹. FIG. 1.3C illustrates particle trajectories of8.0 μm non-magnetic beads at the flow rate of 100 μL min⁻¹. A ferrofluidwith its concentration of 0.049% (v/v) was used; the magnetic fieldgradient was fixed at 132 T m⁻¹. Scale bar: 200 μm. FIG. 1.3D showsthat, in absence of magnetic fields, all of the beads (20.3 and 8.0 μmnon-magnetic beads, and 11.8 μm magnetic beads) were randomlydistributed in the channel. Scale bar: 200 μm. FIG. 1.3E illustratesthat, when magnetic fields were present, non-magnetic beads (20.3 and8.0 μm) will be collected from outlet 1. Most of the magnetic beads(11.8 μm) will be collected at outlet 3, and the rest can be identifiedin outlet 2. Scale bar: 200 μm. FIG. 1.3F is an image of collected beadsfrom 3 outlets. The red fluorescent signal comes from 8.0 μmnon-magnetic beads and the yellow fluorescent signal comes from 11.8 μmmagnetic beads. Scale bar: 200 μm.

FIGS. 1.4A-1.4C demonstrate size of cancer cell lines and labellingresults of WBCs. FIG. 1.4A illustrates the size distribution ofdifferent cancer cell lines—Prostate (PC-3), Breast (MCF-7, MDA-MB-231,HCC1806) and Lung (H1299, H3122, H69, DMS79), and white blood cells fromhealthy donor. FIG. 1.4B illustrates the number of magnetic beads(dynabeads) per white blood cell (n=1000). The average is 34±11dynabeads per WBC. Insect is a WBC labelled with 45 beads. FIG. 1.4Cillustrates the percentage of magnetic content in labelled WBCs. Thevolume fraction of magnetic content in dynabeads is 11.5%, the magneticcontent for labelled WBC was calculated based on volume fraction.

FIGS. 1.5A-1.5F show iFCS applied to cancer cells separation. Certainnumbers of PC-3 prostate cancer cells and 1×10⁶ WBCs were spiked into 1mL ferrofluid with concentration of 0.049% (v/v). The cell mixture wasprocessed at the flow rate of 100 μL min⁻¹; the magnetic field gradientwas fixed at 132 T m⁻¹. FIG. 1.5A shows bright field and fluorescentimages of 1×10⁵ PC-3 cancer cells and WBCs separation process. Greensignal comes from PC-3 cancer cells and red signal comes from WBCs Scalebar: 500 μm. FIG. 1.5B illustrates collected PC-3 from outlet 1. FIG.1.5C shows normalized size distributions of spiked and collected cancercells from outlet 1 for PC-3, MCF-7 and MDA-MB-231. FIG. 1.5D showsmagnetic beads enumeration on collected WBCs from outlet 1 withdifferent concentration of ferrofluid. FIG. 1.5E illustrates recoveryrate and purity for different cancer cell lines (˜100 cells per mL).Recovery rate of 97.9±1.0%, 97.6±1.0%, 98.8±1.4%, 99.4±0.6%, 98.7±0.6%,95.0±1.2%, 95.9±1.3% and 99.7±0.6% were achieved for MCF-7, MDA-MB-231,HCC1806, H1299, H3122, DMS79, H69 and PC-3 cell lines, respectively. Thecorresponding purities are 212.0±1.3%, 23.5±0.7%, 25.2±1.5%, 23.1±0.9%,22.2±0.9%, 24.2±1.7%, 21.7±0.8% and 23.3±0.4%, respectively. Error barsindicate standard deviation, n=3. FIG. 1.5F illustrates a series ofexperiments with different number of spiked PC-3 cancer cells (100, 250,500, 1000 and 2000). An average recovery rate of 98.8% (linear fit,R²=0.9998) was achieved for PC-3 cancer cells.

FIGS. 1.6A-1.6C illustrate an embodiment of an iFCS device design andoperating principle. FIG. 1.6A is a top-view of the FCSv2 device withlabels of inlets, debris filters and outlets. The arrow indicates thedirection of magnetic field during device operation. The deviceintegrates 3 stages into one single device for biomarker- andsize-independent CTC enrichment. Stage one filters out large celldebris. As illustrated in FIG. 1.6B, stage 2 depletes the unboundmagnetic beads and WBCs bound with beads into waste outlet 1 (FIG. 1.6Bii). FIG. 1.6B iii illustrates that stage 3 continuously deflectsunlabeled CTCs into collection outlet, while at the same time focusesWBCs bound with bead into waste outlet 2. FIG. 1.6C is a digital imageof the embodiment of the microfluidic device illustrated in FIG. 1.6Asandwiched between device holders, which include top and bottom magnetarrays repelling each other and are secured with screws and nuts. Thethickness of channel is 250 μm. The width of stage 2 is 1600 μm andwidth of stage 3 is 1200 μm.

FIG. 1.7 illustrates an embodiment of a microfluidic device of thepresent disclosure sandwiched between the two device holders, whichinclude top and bottom magnet arrays repelling each other and securedwith screws and nuts. The center of the stage 3's microchannel (see FIG.1.6A) is aligned exactly with the center of the magnet arrays.

FIG. 1.8 illustrates a simulation result demonstrating that the magneticfield is stronger in the center of the stage 3 region of the deviceillustrated in FIG. 1.6A. As a result, the magnetically labeled whiteblood cells and free magnetic beads are focused in the center of thechannel and are collected through the waste outlet. At the same time,any unlabeled cells, including circulating tumor cells are deflectedinto two sides of the channel and are collected through collectionoutlets. Whole blood is labeled and lysed by RBC lysing buffer before itis processed with the FCSv2. Leukocyte-specific biotinylated antibodies(anti-CD45, anti-CD66b and anti-CD16) and magnetic Dynabeads are addedto the blood sample for incubation.

FIG. 1.9A illustrates the recovery rate of separated cancer cells fordifferent cancer cell lines at the flow rate of 12 mL/h. recovery ratesof 98.46±0.50%, 99.68±0.46%, 99.05±0.75%, 99.35±0.46%, 99.40±0.85%,99.13±0.49%, 99.11±1.25%, and 99.11±0.74% are achieved for HCC1806(breast cancer), HCC70 (breast cancer), MCF7 (breast cancer), MDA-MB-231(breast cancer), H1299 (non-small cell lung cancer), H3122 (non-smallcell lung cancer), DMS79 (small cell lung cancer), and H69 (small celllung cancer) cell lines, respectively. FIG. 1.9B illustrates a series ofspike-in separation experiments in which a certain number (20, 50, 100,and 200) of HCC70 breast cancer cells are spiked into 1 mL of labeledwhite blood cells. An average recovery rate is 99.08%.

FIGS. 1.10A-1.10B illustrate bright field and fluorescence image ofspiked MDA-MB-231 breast cancer cells (labeled with CellTracker Green)during the separation process confirmed the cancer cell trajectories atthe end of stages 2 and 3 in FIGS. 1.10A-1.10C. Scale bars: 500 μm.

FIGS. 2.1A-2.1D illustrate an integrated ferrohydrodynamic cellseparation (iFCS) system and its working principle. FIG. 2.1A (top) is aschematic of an unlabeled circulating tumor cell (CTC) experiencing“diamagnetophoresis” in a colloidal magnetic nanoparticle suspension(ferrofluids) and moving towards the minima of a non-uniform magneticfield. Magnetization of the unlabeled CTCs M_(CTC) is near zero and lessthan its surrounding ferrofluids M_(fluid). The diamagnetic body forceon the cell is generated from magnetic nanoparticle induced pressureimbalance on the cell surface, and is proportional to cell volume. FIG.2.1A (bottom) is a schematic of a magnetic bead labeled white blood cell(WBC) experiencing both “diamagnetophoresis” from its cell surface and“magnetophoresis” from its attached beads in a ferrofluid and movingtowards the maxima of a non-uniform magnetic field due to the fact that“magnetophoresis” outweighs “diamagnetophoresis”. Magnetization of theWBC-bead conjugates M_(WBC-bead) is larger than its surroundingferrofluid medium M_(fluid). Color bar indicates relative amplitude ofthe magnetic field. Red arrows show the direction of cell movement,small black arrows on cell surface show the direction of magneticnanoparticle induced surface pressure on cells, while white arrows showthe magnetophoretic force on magnetic beads. FIG. 2.1B illustrates twoenrichment stages were integrated into a single iFCS device to achievecell size variation-inclusive and tumor antigen-independent enrichmentof viable CTCs, and simultaneous depletion of contaminating WBCs. Priorto device processing, WBCs in blood were labeled with magneticmicrobeads through leukocyte surface biomarkers so that the overallmagnetization of the WBC-bead conjugates was larger than surroundingferrofluids. Magnetization of the unlabeled CTCs was less thanferrofluids. In the first stage, a magnetic field gradient was generatedto push unlabeled and sheath-focused CTCs to remain at the upperboundary of a microchannel, while attract unbound magnetic microbeadsand WBCs labeled with ≥3 microbeads towards a waste outlet. Asignificant percentage of magnetic beads and WBCs were depleted beforethe second stage to alleviate potential bead aggregation. In the secondstage, a symmetric magnetic field with its maximum at the middle of thechannel was used to attract remaining WBC-bead conjugates towards to thechannel center for fast depletion, and direct unlabeled CTCs towards theupper and lower boundaries for collection. Green arrows with gradientsindicate the distribution of magnetic fields in each stage. FIG. 2.1Cillustrates a top-view of an embodiment of an iFCS microchannel. Themicrochannel includes a filter that removes large than ˜50 μm debris, afirst and second stage for CTC enrichment and WBC depletion. FIG. 2.1Dis a digital image of prototype microchannel (left) and assembled iFCSdevice with four permanent magnets in quadrupole configuration inside aholder (right). The microfluidic device and permanent magnets wereplaced within an aluminum manifold during its operation. Scale bars: 1cm.

FIGS. 2.2A-2.2E illustrate system optimizations of an embodiments ofiFCS devices for high recovery (>99%) of viable and rare CTCs (down to˜10 cells mL⁻¹) with low WBC contamination (˜500 cells mL⁻¹) at 12 mLh⁻¹ throughput. FIG. 2.2A illustrates optimization of magnetic fluxdensity and its gradient in microchannels. Using four permanent magnetsin a quadrupole configuration shown here, maximal flux density of up to0.6 T in the first stage (top), and up to 1.5 T in the second stage(bottom) in x-y plane (z=0) were obtained. Cell trajectories shows thatin the first stage WBCs (11.7 μm in diameter) labeled with ≥3 beads andunbound magnetic beads were continuously depleted into a waste outlet,while CTCs (15 μm in diameter) moved to the second stage. In the secondstage, remaining WBCs labeled with <3 beads were further depleted,leaving CTCs at both upper and lower channel walls for collection. Cellflow rate of 200 μL min⁻¹ was used for simulation. Maximal magnetic fluxdensity gradient is 256 T m⁻¹ in the first stage (top) and 625 T m⁻¹ inthe second stage (bottom) in y-z plane (x=0) were obtained. Schematic ofmagnetic ({right arrow over (F)}_(m)) and hydrodynamic drag ({rightarrow over (F)}_(d)) forces on cells and their moving direction (whitearrows; endpoints of white arrows indicting the equilibrium/finalpositions of cells) are overlaid on top of magnetic flux density plots.FIG. 2.2B illustrates optimization of magnetic bead functionalization ofWBCs. Top: distribution of number of magnetic Dynabeads per WBC(n=1000). On overage 34±11 Dynabeads are conjugated onto a single WBC.Inset is a WBC labeled with multiple Dynabeads. Scale bar: 10 μm.Bottom: Magnetic content in labeled WBCs. More than 99.9% of WBCs arelabeled with at least one bead, resulting in a 0.026% volume fraction ofmagnetic materials. This percentage value was used in subsequentoptimization of ferrofluid concentration in order to minimize WBCcontamination at device's outlets. FIG. 2.2C Optimizations of CTCrecovery and WBC depletion (proportional to separation distance ΔY) wereconducted on parameters including ferrofluid concentration (top) anddevice throughput (bottom). An optimal ferrofluid concentration is foundto be 0.028%, while the optimal throughput to process clinicallyrelevant amount of blood is 200 μL min⁻¹. In this optimization, magneticflux density and its gradient are the same as in a, beadfunctionalization of WBCs is the same as in FIG. 2.2B. FIG. 2.2DVisualization of CTC and WBC distributions at the end of microchannelsin the first (top) and second (bottom) stages. CTCs were given a sizerange of 3-32 μm in diameter, while WBCs were given a size range of 5-25μm in diameter. After the first stage, the majority of WBCs weredepleted while all CTCs, regardless of their sizes, moved to the secondstage. After the second stage, all CTCs were collected with a minimalnumber of WBCs contamination/carryover. Yellow areas indicate eithertransfer channel to the second stage or the collection outlets, whilewhite areas indicate waste outlets. FIG. 2.2E illustrates quantificationof CTC and WBC distributions at the end of microchannels in the first(top) and second (bottom) stages. Results show that 96.35% of initialWBCs were depleted after the first stage while all CTCs are preserved,including CTCs that are as small as 3 μm in diameter (top). After thesecond stage, 3.6% of initial WBCs were further depleted and still allCTCs are preserved (bottom). Overall, after two stages, 99.95% of WBCsare depleted from initial samples and all CTCs are preserved. Simulationparameters of d and e include: cell flow rate of 200 μL min⁻¹,ferrofluid with concentration of 0.028%, magnetic flux density of 0.64 Tand 1.5 T for stage I and stage II, flux density gradient of 256 T m⁻¹and 625 T m⁻¹ for stage I and stage II.

FIGS. 2.3A-2.3J illustrate validation of prototype iFCS devices usingcultured cancer cells spiked into WBCs, for over 99% of cancer cellrecovery with minimal WBC contamination (˜500 cells mL⁻¹) at clinicallyrelevant spike ratio (down to ˜10 cells mL⁻¹) and throughput (12 mLh⁻¹). FIG. 2.3A illustrates visualization of cancer cell enrichment andWBCs depletion (top: bright field; bottom: epifluorescence). In thefirst stage of the device, magnetic force attracted labeled WBCs andunbound beads toward waste outlet 1, while unlabeled cancer cells movedcontinuously into the second stage. Cancer cells were labeled with greenfluorescence. Scale bar: 500 μm. FIG. 2.3B demonstrates that, in thesecond stage, magnetic force deflected unlabeled cancer cells from cellmixture toward upper and lower collection outlets. At the same time,labeled WBCs were focused into the middle of the channel and depletedinto waste outlet 2. Top: bright field; bottom: epifluorescence. Scalebar: 500 μm. Dashed lines in fluorescent images indicate the boundariesof the microchannel. FIG. 2.3C illustrates spike-in results from iFCSdevices show high recovery (99.18%) of cancer cells. A series ofspike-in enrichment experiments in which a certain number (10, 25, 50,100, and 200) of HCC70 breast cancer cells were spiked into 1 mL oflabeled WBCs to emulate clinically relevant CTC concentration at acell-processing throughput of 12 mL h⁻¹. An average recovery rate of99.18% was achieved (R²=0.9999, n=3). FIG. 2.3D illustrates sizedistribution of 8 cancer cell lines and WBCs. Both cancer cells and WBCsare polydispersed with overlapping sizes, highlighting the need of iFCSdevelopment to enrichment CTCs in an antigen-independent and sizeinclusive manner. Mean diameter and standard deviations are listed inTable 2.2. FIG. 2.3E is a graph illustrating recovery rates of spikedcancer cells (˜100 cells per mL) from the cancer cell lines, includingtwo small cell lung cancer (SCLC) lines, at a flow rate of 12 mL h⁻¹.Recovery rates of 98.46±0.50%, 99.68±0.46%, 99.05±0.75%, 99.35±0.46%,99.40±0.85%, 99.13±0.49%, 99.11±1.25%, and 99.11±0.74% were achieved forHCC1806, HCC70, MCF7, MDA-MB-231, H1299, H3122, DMS79 (SCLC), and H69(SCLC) cell lines, respectively (n=3). FIG. 2.3F illustrates sizedistribution of spiked and recovered cancer cells after iFCS process,conducted in a single stage iFCS device. iFCS was able to preservecancer cells of all sizes. Mean diameter and standard deviations ofspiked and recovered cancer cells are listed in Table. 2.3. Inset:recovered PC-3 prostate cancer cells showed polydispersity in diameters.Smallest recovered PC-3 cells had a diameter of 6.64 μm. Scale bar: 20μm. FIG. 2.3G illustrates short-term cell viability comparison beforeand after the iFCS process. Cell viability of HCC1806 breast cancercells before and after enrichment is determined to be 98.30±0.56% and97.69±0.70%, with little change. FIG. 2.3H shows representative imagesof Live/Dead staining for before (left) and after (right) enrichment.Calcein AM (green, live cells) and EhD-1 (red, dead cells) channels weremerged. Scale bar: 100 μm. FIG. 2.3I shows representative images ofcultured HCC1806 breast cancer cells after enrichment on 3^(rd)d day. ALive/Dead staining of the cultured cells on day 3 shows excellent cellviability. Scale bar: 100 μm. FIG. 2.3J shows immunofluorescence imagesof an intact spiked HCC1806 cancer cell (left panel) and an intact whiteblood cell conjugated with multiple magnetic beads (right panel). Threechannels including CK (green), CD45 (red), and DAPI (blue) were used.Scale bar: 10 μm. All error bars indicate s.d., n=3.

FIGS. 2.4A-2.4C illustrate variation in CTC sizes and heterogeneity ofCTC surface antigen expressions from breast cancer patient samples(first cohort, n=3). FIG. 2.4A represent bright field andimmunofluorescent images of 7 selected individual CTCs enriched from 3breast cancer (BrC) patients. Five channels were used inimmunofluorescent staining, including leukocyte marker CD45 (red),epithelial CTC marker EpCAM (green), mesenchymal CTC markers N-cadherin(N-cad, cyan) and vimentin (Vim, magenta), and nucleus marker DAPI(blue). White blood cells were identified asCD45+/EpCAM−/N-cad−/Vim−/DAPI+, while CTCs were identified as eitherEpCAM+/CD45−/DAPI+ (epithelial positive), or N-cad+/Vim+/CD45−/DAPI+(mesenchymal positive), or EpCAM+/N-cad+/Vim+/CD45−/DAPI+ (bothepithelial and mesenchymal positive). Scale bar: 10 μm. FIG. 2.4B is agraph representing quantitative analysis of the effective diameter(maximum feret diameter of cells from their bright field images) ofindividual CTCs and WBCs enriched from 3 breast cancer patients'samples. Randomly selected CTCs from these patients revealed a highpolydispersity of cell sizes. CTCs from patient 1 (breast cancer, stageIIIA, BrC-P1-Opt) had diameters of 11.99±7.87 μm (n=24; mean±s.d.;smallest 4.95 μm; largest 33.11 μm); CTCs from patient 2 (breast cancer,stage IA, BrC-P2-Opt) had diameters of 13.73±6.76 μm (n=26; mean±s.d.;smallest 6.00 μm; largest 32.10 μm); CTCs from patient 3 (breast cancer,stage IA, BrC-P3-Opt) had diameter of 9.67±3.60 μm (n=30; mean±s.d.;smallest 4.51 μm; largest 23.48 μm). WBCs pooled from 3 breast cancerpatients had diameter of 9.83±2.27 μm (n=60; mean±s.d.; smallest 5.48μm; largest 21.45 μm) FIG. 2.4C is a graph representing analysis ofsurface antigens expression of individual CTCs from 3 breast cancerpatients' samples revealed a high heterogeneity of epithelial andmesenchymal characteristics in these cells. Cells from each patient aregrouped into three categories (columns): epithelial positive (E+:EpCAM+/CD45−/DAPI+), mesenchymal positive (M+: N-cad+/Vim+/CD45−/DAPI+),both epithelial and mesenchymal positive (E+/M+:EpCAM+/N-cad+/Vim+/CD45−/DAPI+). Numbers in each column indicate theabsolute number of cells in each category. For BrC-P1-Opt, 12.93% ofCTCs was epithelial positive, 78.45% of CTCs was mesenchymal positive,and 8.62% was both epithelial and mesenchymal positive. For BrC-P2-Opt,54.88% of CTCs was epithelial positive, 30.49% of CTCs was mesenchymalpositive, and 14.63% was both epithelial and mesenchymal positive. ForBrC-P3-Opt, 31.75% of CTCs was epithelial positive, 65.08% of CTCs wasmesenchymal positive, and 3.17% was both epithelial and mesenchymalpositive.

FIGS. 2.5A-2.5C illustrate variation in CTC sizes and heterogeneity ofCTC surface antigen expressions from lung cancer patient samples (secondcohort, n=3). FIG. 2.5A represents bright field and immunofluorescentimages of 7 selected individual CTCs enriched from 3 lung cancer (LC)patients. Five channels were used in immunofluorescent staining,including leukocyte marker CD45 (red), epithelial CTC marker CK (green),mesenchymal CTC markers N-cadherin (N-cad, cyan) and vimentin (Vim,magenta), and nucleus marker DAPI (blue). White blood cells wereidentified as CD45+/CK−/N-cad−/Vim−/DAPI+, while CTCs were identified aseither CK+/CD45−/DAPI+ (epithelial positive), or N-cad+/Vim+/CD45−/DAPI+(mesenchymal positive), or CK+/N-cad+/Vim+/CD45−/DAPI+ (both epithelialand mesenchymal positive). Scale bar: 10 μm.

FIG. 2.5B is a graph illustrating quantitative analysis of the effectivediameter (maximum feret diameter of cells from their bright fieldimages) of individual CTCs and WBCs enriched from 3 lung cancerpatients' samples. Randomly selected CTCs from these patients revealed ahigh polydispersity of cell sizes. CTCs from patient 1 (NSCLC, stage IV,LC-P1-Opt) had diameters of 9.73±3.11 μm (n=39; mean±s.d.; smallest 4.59μm; largest 18.52 μm); CTCs from patient 2 (SCLC, stage IV, LC-P2-Opt)had diameters of 10.98±3.41 μm (n=43; mean±s.d.; smallest 5.61 μm;largest 21.13 μm); CTCs from patient 3 (SCLC, stage IV, LC-P3-Opt) haddiameter of 9.23±3.67 μm (n=59; mean±s.d.; smallest 4.55 μm; largest21.67 μm). WBCs from 3 lung cancer patients had diameter of 10.58±2.27μm (n=74; mean±s.d.; smallest 6.86 μm; largest 16.83 μm) FIG. 2.5Cillustrates analysis of surface antigens expression of individual CTCsfrom 3 lung cancer patients' samples revealed a high heterogeneity ofepithelial and mesenchymal characteristics in these cells. Cells fromeach patient are grouped into three categories (columns): epithelialpositive (E+: CK+/CD45−/DAPI+), mesenchymal positive (M+:N-cad+/Vim+/CD45−/DAPI+), both epithelial and mesenchymal positive(E+/M+: CK+/N-cad+/Vim+/CD45−/DAPI+). Numbers in each column indicatethe absolute number of cells in each category. For LC-P1-Opt, 11.84% ofCTCs was epithelial positive, 78.95% of CTCs was mesenchymal positive,and 9.21% was both epithelial and mesenchymal positive. For LC-P2-Opt,46.04% of CTCs was epithelial positive, 50.99% of CTCs was mesenchymalpositive, and 2.97% was both epithelial and mesenchymal positive. ForLC-P3-Opt, 27.93% of CTCs was epithelial positive, 61.71% of CTCs wasmesenchymal positive, and 10.36% was both epithelial and mesenchymalpositive.

FIGS. 2.6A-2.6G illustrate correlation between clinical stages, growthrates of CTC culture, Oncotype Dx scores and heterogeneity of CTCsurface antigen expressions in early stage breast cancer patients (thirdcohort, n=6) FIG. 2.6A represents bright field and immunofluorescentimages of 3 representative CTCs and 1 WBC enriched from breast cancerpatients. Cells were subjected to multiplexed immunofluorescenceassessment with cell-type specific markers detected with distinctwavelength channels, including leukocyte marker CD45 (blue), epithelialCTC marker EpCAM (green), mesenchymal CTC marker vimentin (Vim, red).White blood cells were identified as CD45+/EpCAM−/Vim−, while CTCs wereidentified as EpCAM+/Vim−/CD45− (epithelial positive), EpCAM−/Vim+/CD45−(mesenchymal positive), or EpCAM+/Vim+/CD45− (both epithelial andmesenchymal positive). Scale bar: 10 μm. FIG. 2.6B is a graphillustrating relative counts of CTC subtypes by surface antigenexpression from 6 breast cancer patients' samples. Cells from eachpatient are grouped into three categories (columns): epithelial positive(E+: EpCAM+/Vim−/CD45−), mesenchymal positive (M+: EpCAM−/Vim+/CD45−),both epithelial and mesenchymal positive (E+/M+: EpCAM+/Vim+/CD45−).Numbers in each column indicate the absolute number of cells in eachcategory. FIG. 2.6C illustrates proportional components of CTC profilesfor breast cancer patients, Oncotype Dx scoring indicated for eachpatient. Scores for P2, P3 and P4 patients are not available becausetheir clinical stages are either too high (*confirmed metastasis) or toolow (**non-invasive carcinoma/DCIS). FIG. 2.6D is a graph illustratingthe average proportional components of CTC subtypes are shown withrespect to clinical staging. FIG. 2.6E are representative images ofprimary cell culture of iFCS device output over a 72-hour period. NCCNstaging for each BrC patient is represented next to the patientdesignation. Scale bar: 200 μm. FIG. 2.6F is a graph illustratingcombined total number of cells quantified from primary cell cultureimaging for each BrC patient at 0 hours and the change in counts at 72hours. FIG. 2.6G illustrates growth rate of cells in primary culture foreach BrC patient ((cell number at 72 hr−cell number at 0 hr)/cell numberat 72 hr).

FIGS. 3.1A-3.1B illustrate magnetic content of cells at various stagesin an embodiment of a device of the present disclosure. FIG. 3.1Aillustrates magnetic content of undeflected (left) and deflected (right)white blood cells (WBCs) at stage I. FIG. 3.1B illustrates magneticcontent of undeflected (left) and deflected (right) WBCs at stage II.

FIG. 3.2 is a graph illustrating the average number of Dynabeads perwhite blood cell (WBC) after cell enrichment. The device carried over onaverage 533±34 WBCs per 1 milliliter of blood processed. Much of thecarryover was derived from WBCs that were either not labeled or labeledwith just one magnetic bead.

FIG. 3.3 illustrates the size distribution of clinical CTCs fromnon-small cell lung cancer (NSCLC) patients, small cell lung cancer(SCLC) patients, and breast cancer (BrC) patients. Details are listed inTable 2.4.

FIG. 3.4 are images of cytopathological staining of spiked HCC1806cancer cells after enrichment. Scale bars: 20 μm.

FIG. 3.5A shows bright field and immunofluorescent images of CTCsenriched from breast cancer patient. Five channels were used inimmunofluorescent staining, including leukocyte marker CD45 (red),epithelial CTC marker EpCAM (green), mesenchymal CTC markers N-cadherin(N-cad, cyan) and vimentin (Vim, magenta), and nucleus marker DAPI(blue). FIG. 3.5B illustrate bright field and immunofluorescent imagesof CTC enriched from lung cancer patients. Five channels were used inimmunofluorescent staining, including leukocyte marker CD45 (red),epithelial CTC marker CK (green), mesenchymal CTC markers N-cadherin(N-cad, cyan) and vimentin (Vim, magenta), and nucleus marker DAPI(blue). Scale bars: 10 μm.

FIGS. 3.6A-3.6C illustrate correlation of the numbers of each CTCsubtype with tumor grade in third cancer patient cohort.

FIG. 3.7A illustrates magnetization of the as-synthesized ferrofluid.Solid lines are the fitting of the experimental date to the Langevinfunction. Saturation magnetization of this ferrofluid was 0.104 kA m⁻¹,corresponding to a 0.028% volume fraction or concentration. FIG. 3.7B isa rheological plot of the ferrofluid. The viscosity of ferrofluid wasmeasured to be 0.99 mPa·s. FIG. 3.7C is a transmission electronmicroscopy (TEM) image of the maghemite nanoparticles. Scale bar: 10 nm.

FIG. 3.8 is a schematic illustration of a magnet array according to anembodiment of multi-stage microfluidic device of the present disclosureshowing orientation of the magnet array relative to a first separationstage and second separation stage of the device.

DETAILED DESCRIPTION

In various aspects, microfluidic devices and methods of usingmicrofluidic devices are provided for separating and/or enrichingcirculating tumor cells (CTCs) in a biological sample such as wholeblood. The methods do not involve labeling of the CTCs and are capableof high throughputs with high levels of retention and separation of thecirculating tumor cells.

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. The skilled artisan will recognize many variants andadaptations of the embodiments described herein. These variants andadaptations are intended to be included in the teachings of thisdisclosure and to be encompassed by the claims herein.

All publications and patents cited in this specification are cited todisclose and describe the methods and/or materials in connection withwhich the publications are cited. All such publications and patents areherein incorporated by references as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. Such incorporation by reference is expressly limited tothe methods and/or materials described in the cited publications andpatents and does not extend to any lexicographical definitions from thecited publications and patents. Any lexicographical definition in thepublications and patents cited that is not also expressly repeated inthe instant specification should not be treated as such and should notbe read as defining any terms appearing in the accompanying claims. Thecitation of any publication is for its disclosure prior to the filingdate and should not be construed as an admission that the presentdisclosure is not entitled to antedate such publication by virtue ofprior disclosure. Further, the dates of publication provided could bedifferent from the actual publication dates that may need to beindependently confirmed.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of biochemistry, molecular biology, microfluidics,and the like, which are within the skill of the art. Such techniques areexplained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice or testing of thepresent disclosure, the preferred methods and materials are nowdescribed. Functions or constructions well-known in the art may not bedescribed in detail for brevity and/or clarity.

It should be noted that ratios, concentrations, amounts, and othernumerical data can be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a numerical range of “about 0.1%to about 5%” should be interpreted to include not only the explicitlyrecited values of about 0.1% to about 5%, but also include individualvalues (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%,2.2%, 3.3%, and 4.4%) within the indicated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, e.g. thephrase “x to y” includes the range from ‘x’ to ‘y’ as well as the rangegreater than ‘x’ and less than ‘y’. The range can also be expressed asan upper limit, e.g. ‘about x, y, z, or less’ and should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘less than x’, less than y′, and ‘less than z’.Likewise, the phrase ‘about x, y, z, or greater’ should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘greater than x’, greater than y′, and ‘greaterthan z’. In some embodiments, the term “about” can include traditionalrounding according to significant figures of the numerical value. Inaddition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numericalvalues, includes “about ‘x’ to about ‘y’”.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. It will be further understoodthat terms, such as those defined in commonly used dictionaries, shouldbe interpreted as having a meaning that is consistent with their meaningin the context of the specification and relevant art and should not beinterpreted in an idealized or overly formal sense unless expresslydefined herein.

The articles “a” and “an,” as used herein, mean one or more when appliedto any feature in embodiments of the present invention described in thespecification and claims. The use of “a” and “an” does not limit themeaning to a single feature unless such a limit is specifically stated.The article “the” preceding singular or plural nouns or noun phrasesdenotes a particular specified feature or particular specified featuresand may have a singular or plural connotation depending upon the contextin which it is used.

As used herein, “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps, or components, or groups thereof.Moreover, each of the terms “by”, “comprising,” “comprises”, “comprisedof,” “including,” “includes,” “included,” “involving,” “involves,”“involved,” and “such as” are used in their open, non-limiting sense andmay be used interchangeably. Further, the term “comprising” is intendedto include examples and aspects encompassed by the terms “consistingessentially of” and “consisting of.” Similarly, the term “consistingessentially of” is intended to include examples encompassed by the term“consisting of”.

In this disclosure, “consisting essentially of” or “consistsessentially” or the like, when applied to methods and compositionsencompassed by the present disclosure refers to compositions like thosedisclosed herein, but which may contain additional structural groups,composition components or method steps (or analogs or derivativesthereof as discussed above). Such additional structural groups,composition components or method steps, etc., however, do not materiallyaffect the basic and novel characteristic(s) of the compositions ormethods, compared to those of the corresponding compositions or methodsdisclosed herein. “Consisting essentially of” or “consists essentially”or the like, when applied to methods and compositions encompassed by thepresent disclosure have the meaning ascribed in U.S. patent law and theterm is open-ended, allowing for the presence of more than that which isrecited so long as basic or novel characteristics of that which isrecited is not changed by the presence of more than that which isrecited, but excludes prior art embodiments.

As used herein, “about,” “approximately,” “substantially,” and the like,when used in connection with a numerical variable, can generally refersto the value of the variable and to all values of the variable that arewithin the experimental error (e.g., within the 95% confidence intervalfor the mean) or within +/−10% of the indicated value, whichever isgreater. As used herein, the terms “about,” “approximate,” “at orabout,” and “substantially” can mean that the amount or value inquestion can be the exact value or a value that provides equivalentresults or effects as recited in the claims or taught herein. That is,it is understood that amounts, sizes, formulations, parameters, andother quantities and characteristics are not and need not be exact, butmay be approximate and/or larger or smaller, as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art such thatequivalent results or effects are obtained. In some circumstances, thevalue that provides equivalent results or effects cannot be reasonablydetermined. In general, an amount, size, formulation, parameter or otherquantity or characteristic is “about,” “approximate,” or “at or about”whether or not expressly stated to be such. It is understood that where“about,” “approximate,” or “at or about” is used before a quantitativevalue, the parameter also includes the specific quantitative valueitself, unless specifically stated otherwise.

As used herein, the terms “optional” or “optionally” indicates that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, “kit” refers to a collection of at least two componentsconstituting the kit. Together, the components constitute a functionalunit for a given purpose. Individual member components may be physicallypackaged together or separately. For example, a kit comprising aninstruction for using the kit may or may not physically include theinstruction with other individual member components. Instead, theinstruction can be supplied as a separate member component, either in apaper form or an electronic form which may be supplied on computerreadable memory device or downloaded from an internet website, or asrecorded presentation.

As used herein, “instruction(s)” refers to documents describing relevantmaterials or methodologies pertaining to a kit. These materials mayinclude any combination of the following: background information, listof components and their availability information (purchase information,etc.), brief or detailed protocols for using the kit, trouble-shooting,references, technical support, and any other related documents.Instructions can be supplied with the kit or as a separate membercomponent, either as a paper form or an electronic form which may besupplied on computer readable memory device or downloaded from aninternet website, or as recorded presentation. Instructions can compriseone or multiple documents, and are meant to include future updates.

As used herein, “attached” can refer to covalent or non-covalentinteraction between two or more molecules. Non-covalent interactions caninclude ionic bonds, electrostatic interactions, van der Walls forces,dipole-dipole interactions, dipole-induced-dipole interactions, Londondispersion forces, hydrogen bonding, halogen bonding, electromagneticinteractions, π-π interactions, cation-π interactions, anion-πinteractions, polar π-interactions, and hydrophobic effects.

A “biocompatible” substance or fluid, as described herein, indicatesthat the substance or fluid does not adversely affect the short-termviability or long-term proliferation of a target cell within aparticular time range.

Microfluidic Devices & Systems and Methods of Use Thereof

In various aspects, microfluidic devices, systems, kits, and methods ofusing multi-stage microfluidic devices are provided for high throughputsorting, separation, and/or enrichment of circulating tumor cells (CTCs)and other unlabeled rare cells in a biological sample such as blood,where the CTCs or other rare cells do not need to be labeled.

Although microfluidics-based methods have been explored as a new avenueto enrich and study CTCs for the past decade, previous approaches werebased on the use of specific tumor antigens (marker-dependent) or cellsize threshold (cell size-dependent) for enrichment, and suffereddisadvantages due to limitations of these approaches. For example,marker-dependent methods that relied on EpCAM or other combination oftumor cell surface antigens were rendered ineffective due to inherentheterogeneity of tumor subtypes. The significant difference amongvarious markers and their expression levels in CTCs undergoing EMT wasdifficult to predict, resulting in incomplete recovery of CTCs fromclinical samples. Cell size-dependent methods, on the other hand, basedon a presumed size difference between blood and cancer cells, provedlargely ineffective because a significant percentage of CTCs incirculation were comparable or smaller than blood cells. Measurements ofwhite blood cells (WBCs) and cultured cancer cells revealed that therewas a significant size overlap between the two, and an appreciablepercentage (e.g., ˜35% for DMS 79 and H69 small cell lung cancer celllines) of cancer cells were smaller than ˜10 μm. In addition, clinicallyisolated CTCs were reported to be as small as ˜6 μm. As a result, fewcell size-dependent methods could achieve complete recovery and low WBCcontamination simultaneously. Furthermore, due to their fragile nature,CTCs need to be processed with gentle enrichment conditions to keeptheir viability and tumorigenic capability for downstream studies. Thus,new devices and methods are needed that can enrich viable CTCsregardless of their surface antigens and size profiles.

In some aspects, a multi-stage microfluidic device is provided forenriching CTCs in a biological sample. The device can include at leastthree stages, although there may be more in some embodiments. Therefore,the terms first, second, third and so-on, when used to describe thestages, should not be considered limiting on the total number of stagesbut is used for simplicity to describe the relative ordering of thestages. Additional stages, not explicitly described, may in some aspectsappear before the first stage.

Embodiments of the present disclosure include a cell separation systemand/or kit for enriching CTCs in a biological sample. In embodiments,the cell separation system/kit can include a plurality of magnetic microbeads adapted for conjugation to white blood cells in the biologicalsample and not to the CTCs, a biocompatible superparamagnetic sheathingfluid or composition including a plurality of magnetic nanoparticles,and a multi-stage microfluidic device for magnetically separating theCTCs from the biological sample. The systems/devices of the presentdisclosure allow enrichment of CTCs are size independent and do notrequire labeling the CTCs.

In embodiments the plurality of magnetic microbeads can include variousbiocompatible magnetic materials, such as, but not limited to iron oxidebased magnetic material (e.g., magnetite (Fe₃O₄), maghemite (Fe₂O₃), orcombinations of these. The magnetic materials for the microbeads shouldbe non-toxic to cells. The magnetic microbeads of the present disclosureare adapted for conjugation with white blood cells (WBCs) such that themicrobeads bind/conjugate with WBCs in the sample and do notsubstantially bind/associate with the CTCs in the sample (e.g., they donot specifically bind/conjugate/associate with the CTCs or othercomponents present in the sample other than WBC's, and any non-specificassociation with CTCs, if present, is insignificant/negligible). Inembodiments the magnetic microbeads are functionalized for specificbinding to WBCs, such as by surface-functionalization with one or morebinding agent(s) for specific binding to WBCs but that will notsubstantially bind the CTCs (e.g., streptavidin/avidin conjugated tobiotinylated white blood cell-specific antibodies). In embodiments themagnetic microbeads comprise streptavidin coated magnetic Dynabeads(Life Technologies, Carlsbad, Calif.). In embodiments, the streptavidincoated magnetic beads are further conjugated to white-blood cellspecific antibodies for specific binding to white blood cells. Examplesof white blood cell-specific antibodies include, but are not limited toanti-CD45, anti-CD66b and anti-CD16, and combinations thereof (whileother antibodies can be used, a combination of the above antibodies willtarget substantially all WBC's present in a blood sample).

In embodiments, the biological sample is whole blood. In embodiments,the sample is whole blood that has been treated with a lysis buffer tolyse/remove red blood cells. In embodiments, the biological sample iscombined with the magnetic microbeads prior to introduction to thedevice of the present disclosure to produce a magnetically labeledbiological sample, such that the WBCs in the sample are substantiallyconjugated to magnetic microbeads prior to introduction. In embodiments,about 95% to 99.9%, or more of the white blood cells are conjugated toone or more magnetic microbeads. In embodiments, a majority of whiteblood cells in the magnetically labeled sample are conjugated to about 1or more magnetic microbeads. In embodiments, a majority of white bloodcells in the magnetically labeled sample are conjugated to about 1 toabout 60 magnetic microbeads. In embodiments, the white blood cells inthe magnetically labeled sample are conjugated to an average of about20-50 magnetic microbeads. As used herein, the terms “magneticallylabeled biological sample” and “magnetically labeled sample” refer toembodiments described above where a biological sample is combined withmagnetic microbeads adapted for specific conjugation with WBC's and notto CTC's or other components of the sample, which are not substantiallyassociated with the magnetic microbeads or otherwise magneticallylabeled. Thus the terms “magnetically labeled biological sample” and“magnetically labeled sample” should not be interpreted to indicate anymagnetic labeling of any other components of the biological sample otherthan the WBCs.

In embodiments, the biocompatible superparamagnetic sheathing fluid(also periodically referred to herein as a “ferrofluid”) of the presentdisclosure is a colloidal suspension of magnetic nanoparticles, coatedby a biocompatible surfactant and suspended in a carrier fluid. Theferrofluid of the present disclosure is biocompatible and non-toxic toCTCs. In embodiments, the magnetic nanoparticles are a non-toxicmagnetic material, such as, but not limited to iron oxide materials(e.g., magnetite (Fe₃O₄), maghemite (Fe₂O₃), and combinations of these.In embodiments, the magnetic nanoparticles are iron oxide particles(e.g., maghemite (Fe₂O₃)). Materials such as iron, cobalt, cobaltferrite, and FePt are potentially toxic to cells, but could potentiallybe used if first rendered biocompatible/nontoxic by biocompatiblecoatings, etc. The magnetic nanoparticles can have a diameter of about1-20 nm. In embodiments they have an average diameter of about 8-12 nm(e.g., about 11 nm). In embodiments, the magnetic nanoparticles arecoated in a biocompatible surfactant to reduce agglomeration and toincrease biocompatibility. In embodiments, the biocompatible surfactantcan include electric double layer surfactant, polymer surfactant,inorganic surfactant, or a combination thereof. In embodiments, thesurfactant is polymethyl methacrylate-polyethylene glycol (PMMA-PEG). Inembodiments, the carrier medium can include biocompatible carrierfluids, such as, but not limited to, water, salt solution, or acombination. In embodiments, the carrier medium is a balanced saltsolution, such as Hank's balanced salt solution (HBSS). In anembodiment, the biocompatible superparamagnetic sheathing fluid includesmaghemite nanoparticles (Fe₂O₃) coated with polymethylmethacrylate-polyethylene glycol (PMMA-PEG) and 10% (v/v) 10× Hank'sbalanced salt solution (HBSS). In embodiments, the pH is about 7, andthe osmotic pressure is close to that of a biological (e.g., human)cell. In embodiments, the ferrofluid concentration (volume fraction ofmagnetic particles) is about 0.01% to 0.04% and intervening ranges. Forinstance, in embodiments the ferrofluid concentration can be about 0.024to 0.033% (v/v). In embodiments the concentration is about 0.028%. Theviscosity of the biocompatible superparamagnetic sheathing fluid variesbased on the concentration of magnetic particles, the surfactant chosen,as well as the carrier fluid. In embodiments, the viscosity of theferrofluid is about 0.95 mPa·s to 1.1 mPa·s. In embodiments of systemsor kits of the present disclosure, the kit/system may include the fullyprepared biocompatible superparamagnetic sheathing fluid as describedabove, and/or a prepared biocompatible superparamagnetic sheathing fluidalong with instructions for diluting the fluid with additional carrierfluid to adjust the concentration/volume fraction of magneticnanoparticles in the sheathing fluid. In embodiments, systems or kits ofthe present disclosure may include a biocompatible superparamagneticsheathing composition that includes the plurality of magneticnanoparticles and instructions for combining the magneticnanoparticles/biocompatible superparamagnetic sheathing composition witha biocompatible surfactant and biocompatible carrier fluid to make abiocompatible superparamagnetic sheathing fluid of the presentdisclosure. In embodiments, systems/kits of the present disclosure caninclude the plurality of magnetic nanoparticles and the biocompatiblesurfactant (separately or mixed (e.g., such that the surfactant coatsthe magnetic nanoparticles) and instructions for combining thebiocompatible superparamagnetic sheathing composition with abiocompatible carrier fluid to make a biocompatible superparamagneticsheathing fluid of the present disclosure.

The multi-stage microfluidic devices of the present disclosure areadapted for use with the biocompatible superparamagnetic sheathingfluids and the magnetic microbeads for enrichment of CTCs from abiological sample. Embodiments of the multi-stage microfluidic devicesinclude a filter section, a first separation stage, and a secondseparation stage (in description of embodiments of the device in partsof the present disclosure, these three sections may also be referred toas a first, second, and third stage).

In embodiments of multi-stage microfluidic devices of the presentdisclosure the filter section can include a first microfluidic channel,a first fluid inlet, and one or more filters along a length of the firstmicrofluidic channel. The first fluid inlet is configured to receive thebiological sample, where the biological sample has been combined withthe magnetic microbeads prior to introduction to the multi-stagemicrofluidic device such that a majority of the white blood cells in thebiological sample are conjugated to one or more magnetic microbeads.This can be referred to as the magnetically labeled sample and/ormagnetically conjugated biological sample. In embodiments, the one ormore filters are configured to remove a first plurality of wasteparticles from the biological sample (e.g., largeparticulate/agglomerated matter in the sample). After the first filtersection, the filtered biological sample proceeds to the first separationstage.

In embodiments of multi-stage microfluidic devices of the presentdisclosure the first separation stage includes a second microfluidicchannel fluidly connected to the first microfluidic channel and having asecond fluid inlet to receive the biocompatible superparamagneticsheathing fluid at a first end, and a first fluid outlet at a secondend. In such embodiments, the second microfluidic channel is configuredto combine the magnetically labeled biological sample from the firstfilter section with the biocompatible superparamagnetic sheathing fluidfrom the second fluid inlet. It is also contemplated that, inembodiments, the biocompatible superparamagnetic sheathing fluid can becombined with the biological sample and the magnetic microbeads prior tointroduction to the device instead of introduced after the filter stagevia a second fluid inlet. Thus, in embodiments, the first separationstage may not include a second fluid inlet. In other embodiments, asecond fluid inlet may be located in the filter section before or afterthe filters. The first separation stage is fluidly connected to thesecond separation stage. The second separation stage includes a thirdmicrofluidic channel fluidly connected to the second microfluidicchannel and having a second fluid outlet and at least a first and secondcirculating tumor cell outlet.

The multi-stage microfluidic devices of the present disclosure alsoinclude one or more magnetic sources adjacent to the multi-stagemicrofluidic device. The magnetic source(s) are configured to produce anon-uniform magnetic field along the second microfluidic channel. Themagnetic field distribution is non-uniform along one or more dimensions(e.g., length, width, and/or height) of the second microfluidic channel.In embodiments, magnetic source(s) are configured to produce anon-uniform magnetic field along the length, width, and/or height of thesecond microfluidic channel. In embodiments, the magnetic field has acomponent sufficiently perpendicular to the length of the secondmicrofluidic channel to cause a plurality of white blood cellsconjugated to the magnetic beads to be deflected into the first fluidoutlet. The non-uniform magnetic field also focuses the CTCs (which arenot magnetically labeled) away from the first fluid outlet and towardthe second separation stage/third microfluidic channel. The magneticsource(s) are also configured to produce a substantially symmetricmagnetic field along the third microfluidic channel, where thesubstantially symmetric magnetic field has a field maximum along alength of the third microfluidic channel sufficient to cause the whiteblood cells conjugated to the magnetic beads in the third microfluidicchannel to be focused toward a center of the third microfluidic channeland to exit the channel via the second fluid outlet and to causecirculating tumor cells to be deflected towards an outer portion of thethird microfluidic channel and to exit the channel via the first and/orsecond circulating tumor cell outlet.

In embodiments, the multi-stage microfluidic device has a serpentineshape. In embodiments, the filter section has a first end and a secondend fluidly connected by the first microfluidic channel, where the firstfluid inlet is fluidly connected to the first microfluidic channel atthe first end, and the one or more filters are located between the firstand second end of the first microfluidic channel. In embodiments, thefirst separation stage has a third end and fourth end fluidly connectedby the second microfluidic channel, where the third end is connected tothe second end of the first microfluidic channel of the filter sectionby a first u-shaped channel, where the second fluid inlet is fluidlyconnected to the second microfluidic channel at the third end and thefirst fluid outlet is fluidly connected to the second microfluidicchannel at the fourth end. In embodiments, the second separation stagehas a fifth end and a sixth end fluidly connected by the thirdmicrofluidic channel, where the fifth end is connected to the fourth endof the second microfluidic channel of the first separation stage by asecond u-shaped channel, where the second fluid outlet is fluidlyconnected to the third microfluidic channel at the sixth end andconfigured to receive materials flowing through a central portion of thethird microfluidic channel. In embodiments, the first and secondcirculating tumor cell outlets are each fluidly connected to the thirdmicrofluidic channel at the sixth end and offset from the center of thechannel and configured to receive material flowing near the outerportion of the channel. In embodiments the first and second u-shapedchannels located between the first filter section and the firstseparation stage and between the first separation stage and secondseparation stage, respectively, provide the serpentine shape to themulti-stage microfluidic device. As used herein, “u-shaped” indicates ageneral u-shape but which may have a variety of configurations (e.g.,rounded, squared, or even angular). In embodiments the U-shape channelhas an angle of curvature of about 120° to about 180°.

In embodiments of the microfluidic device of the present disclosure, thefirst fluid outlet is offset from a central diameter of the secondmicrofluidic channel and configured to receive white blood cellsconjugated to magnetic beads and unconjugated magnetic beads flowing onone side of the second microfluidic channel (e.g., due to deflectionfrom the non-uniform magnetic field produced by the one or more magneticsources).

In embodiments, the magnetic source is a magnet or magnetic array. Inembodiments, the magnetic source is a first magnet array and a secondmagnet array arranged in a quadrupole configuration such that the firstmagnet array and the second magnet array are oriented to repel eachother, one embodiment of which is illustrated in FIG. 3.8. Inembodiments, the second separation stage (e.g., third stage) issandwiched between the first magnet array and the second magnet arrayand oriented such that the length of the third microfluidic channel iscentrally aligned between the first magnet array and the second magnetarray. In embodiments, the magnetic source is located about 800 to 1200μm from the third microfluidic channel. In embodiments, one magneticarray is about 800-1200 μm from the top of the third microfluidicchannel (second separation stage), and the second magnetic array isabout 1000 μm from the bottom of the third microfluidic channel. Inembodiments, the second microfluidic channel of the first separationstage (e.g., second stage) is offset from the magnetic source such thatthe magnetic source provides a non-uniform magnetic field. Inembodiments, the magnetic source is the same first and second magnetarray arranged in a quadrupole configuration sandwiching the thirdmicrofluidic channel, but the second microfluidic channel runs along aside of the magnetic array, such that the second microfluidic channel isnot centrally aligned between the first and second magnet array. Inembodiments, the second and third microfluidic channels aresubstantially parallel; thus there is the same distance above and belowthe channels from the magnetic source as set forth above for the thirdmicrofluidic channel e.g., 800-1200 μm), yet the lateral distance of themagnets from the center of the second microfluidic channel is greater asillustrated in FIG. 3.8.

In embodiments of the microfluidic device of the present disclosure, theone or more magnetic sources produce a flux density of about 0.3-0.8 T(e.g., 0.4 to 0.6 T) in the first separation stage and about 1-1.8 T(e.g., 1.3 to 1.5 T) in the second separation stage. In embodiments, theone or more magnetic sources produce a flux density gradient that rangesfrom about 4 to 256 T m-1 in the first separation stage and from about0-625 T m-1 in the second separation stage, wherein the flux densitygradient is lower towards the center of the third microfluidic channelof the second separation stage than at the outer portion, such that themagnetically conjugated WBC's are deflected toward the second fluidoutlet.

In embodiments, one or more of the first microfluidic channel, thesecond microfluidic channel, and the third microfluidic channel have athickness of about 100 μm to about 500 μm. In embodiments, the thicknessof the microfluidic channels can range from about 100 μm to 500 μm, etc.In embodiments the thickness of the channel is about 300 μm.

In embodiments, the second microfluidic channel has a width of about 500μm to 5000 μm, about 1200 μm to 2000 μm, about 1400 μm to 1800 μm, orabout 1600 μm, and the third microfluidic channel has a width of about500 μm to 5000 μm, about 800 μm to 1600 μm, about 1000 μm to 1400 μm, orabout 1200 μm. In embodiments, the second microfluidic channel has anoptimized width of about 1600 μm, and the third microfluidic channel hasan optimized width of about 1200 μm. The widths of the second and thirdmicrofluidic channels was optimized based on a combination of operatingparameters, including magnetic flux density, operational flow rates,concentration of the superparamagnetic fluid, and the like. Inembodiments, the microfluidic channels can have a length of about of52000-57000 μm.

In embodiments, the second microfluidic channel has an optimized lengthof 55000 μm, with an acceptable range of 52000-57000 μm, and the thirdmicrofluidic channel has an optimized length of 55000 μm, with anacceptable range of 52000-57000 μm.

In embodiments of the microfluidic device of the present disclosure, thecirculating tumor cells that exit via the first and/or secondcirculating tumor cell outlet comprise about 95% or more, 97%, or more,or 99% or more of the total number of circulating tumor cells present inthe biological sample inserted into the first fluid inlet when inoperation.

The present disclosure also includes methods of using the microfluidicdevices of the present disclosure and the systems of the presentdisclosure (e.g., microfluidic device plus biocompatiblesuperparamagnetic sheathing fluid and magnetic microbeads) to enrichCTCs in a biological sample. In some aspects, the methods are capable ofisolating a majority of the unlabeled rare cells. In some aspects, theunlabeled rare cells are circulating tumor cells in a whole bloodsample, and the majority of the circulating tumor cells comprises about90%, about 95%, about 97%, about 99%, or more of the circulating tumorcells as compared to a total number of circulating tumor cells presentin the biological sample inserted into the first fluid inlet when inoperation.

In some aspects, the biological sample includes whole blood, wherein thewhole blood includes a plurality of components. In some aspects, theplurality of components comprises magnetically labelled white bloodcells, and wherein at least 95%, at least 98%, at least 99%, at least99.9%, or more of the white blood cells are not collected in the one ormore circulating tumor cell outlets as compared to a total number ofwhite blood cells present in the whole blood inserted into the firstfluid inlet when in operation. This can mean, for instance, that atleast 95%, at least 98%, at least 99%, at least 99.9%, or more of thewhite blood cells are collected in one or more of the filters, the firstfluid outlet, and the second fluid outlet as compared to a total numberof white blood cells present in the whole blood inserted into the firstfluid inlet when in operation. This can result in, for example, that atleast 90%, 92%, 95%, or more of the unlabeled rare cells are collectedin the one or more circulating tumor cell outlets as compared to a totalnumber of unlabeled rare cells present in the whole blood inserted intothe first fluid inlet when in operation.

Methods are provided for enriching, separating, or isolating unlabeledrare cells such as circulating tumor cells from a sample, e.g. abiological sample such as whole blood. In some aspects, the biologicalsample is or includes whole blood. In some embodiments the biologicalsample includes whole blood treated with a lysis buffer to lyse redblood cells as described above. In some aspects, the biological sampleincludes about 1 to 1000 circulating tumor cells per milliliter of thebiological sample, including both natural samples and samples spikedwith CTCs for research purposes. In embodiments, the biological samplecomprises about 1-10 circulating tumor cells per milliliter of thebiological sample. Examples of the circulating tumor cells can includethose selected from the group consisting of a primary cancer cell, alung cancer cell, a prostate cancer cell, a breast cancer cell, apancreatic cancer cell, and a combination thereof.

The methods can include enriching circulating tumor cells in a sample ofwhole blood, wherein the whole blood includes unlabeled rare cells andwhite blood cells, the method including: (i) adding a plurality ofmagnetic beads to the sample to produce sample having magneticallylabeled WBCs, wherein at least some of the white blood cells areassociated with the magnetic beads; (ii) filtering the magneticallylabeled sample in a microfluidic device to produce a filtered sample byremoving large cell debris from the magnetically labeled sample; (iii)separating at least a portion of the white blood cells that areassociated with the magnetic beads by flowing the filtered samplethrough a sheath flow in a nonuniform magnetic field to produce a firstenriched sample; and (iv) isolating a majority of the unlabeled rarecells by magnetic flow focusing the first enriched sample in amicrofluidic channel.

In embodiments, methods of enriching CTCs in a biological sample of thepresent disclosure include combining the biological sample (e.g., asample from a biological host; e.g., a sample having a plurality ofcomponents) with a plurality of magnetic microbeads adapted to conjugateto white bloods cells such that a majority of white blood cells in thesample become conjugated to at least one magnetic microbead and thenintroducing the magnetically labeled biological sample (including themagnetic microbeads and magnetic microbead-conjugated WBCs) into thefirst fluid inlet of a microfluidic device and combining themagnetically labeled biological sample with a biologically compatiblesuperparamagnetic fluid, introduced via the second fluid inlet, whereinthe magnetically labeled sample and the superparamagnetic fluid areintroduced at a flow rate sufficient to cause the biological sample toflow along the first microfluidic channel, the second microfluidicchannel, and the third microfluidic channel such that a majority of themagnetic microbeads and white blood cells are deflected to the first orsecond fluid outlet and a majority of the circulating tumor cells fromthe biological sample are collected in the one or more circulating tumorcell outlets.

In embodiments, the biological sample is a sample from an animal host(e.g., a mammal, human, etc.). In embodiments, the biological sample isa fluid having a plurality of components (e.g., cells, fluids, etc.)(e.g., blood, urine, saliva, exudate, homogenized tissue in abiocompatible fluid, etc.). In embodiments, the biological sample isfrom a human. In embodiments, the biological sample is whole blood orwhole blood that has been treated with lysis buffer to lyse red bloodcells. In embodiments, the biological sample is whole blood, or RBClysed blood from a human host having cancer or suspected of havingcancer.

In embodiments of methods of the present disclosure, the flow rate ofthe fluids/samples (e.g., the magnetically labeled biological sample,the combined magnetically labeled sample and superparamagnetic fluid,etc.) in the microfluidic device is about 10-400 microliters per minute,or any intervening range. In embodiments, the flow rate is 10-250microliters per minute. In some embodiments, the flow rate is about 0.6to 24 milliliters of the fluids per hour, and intervening ranges, e.g.,about 0.6 to 15 ml/hr.

Untreated biological samples have various amounts of circulating tumorcells, dependent on the type of cancer, the stage of cancer, othercancer treatments, etc. In general, un-spiked samples from a host haveabout 1-10 circulating tumor cells/ml of the biological sample. However,for research purposes, sometimes samples are spiked with additionalCTCs. Also the level of CTCs can be higher depending on the tumor gradeof a host. Thus, in embodiments, the biological sample can have fromabout 1 to about 1000 circulating tumor cells/ml of biological sample.It will be understood that for research purposes even higher amounts ofcirculating tumor cells may be present in a sample and still be withinthe scope of the present disclosure. However, unlike some previousmethods, the present methods/devices/systems are able to detect/enrichCTCs from a sample having 10 or fewer CTCs/ml of biological sample.

In embodiments, the CTCs can include, but are not limited to, a primarycancer cell, a lung cancer cell, a prostate cancer cell, a breast cancercell, a pancreatic cancer cell, and a combination thereof.

Methods of the present disclosure for enriching CTCs in a sample ofwhole blood (or RBC-lysed whole blood), where the whole blood comprisesunlabeled rare cells and white blood cells, include adding a pluralityof magnetic beads to the sample to produce a magnetically labeledsample, where a majority of the white blood cells are associated withthe magnetic beads and wherein the magnetic beads do not conjugate tothe unlabeled rare cells; filtering the magnetically labeled sample in amicrofluidic device to produce a filtered sample by removing large celldebris from the magnetically labeled sample; separating at least aportion of the white blood cells that are associated with the magneticbeads by flowing the filtered sample through a sheath flow in abiocompatible superparamagnetic sheathing fluid in a nonuniform magneticfield to produce a first enriched sample; and then isolating a majorityof the unlabeled rare cells by flowing the first enriched sample througha channel with a substantially symmetric focused magnetic field suchthat a majority of any remaining white blood cells associated with themagnetic beads are focused towards a center of the channel and to acentral channel outlet and the majority of the unlabeled rare cells arecollected from one or more peripheral channel outlets.

The methods can include introducing the biological sample/magneticallylabeled biological sample, which is preferably combined with thebiocompatible magnetic microbeads prior to introduction, into the firstfluid inlet of a microfluidic device described herein at a flow ratesufficient to cause the biological sample to flow along the microfluidicchannel(s) of the device such that a majority of the circulating tumorcells from the biological sample are collected in the one or morecirculating tumor cell outlets. In embodiments the magnetically labeledbiological sample can also be combined with the superparmagentic fluidprior to introduction into the device. The methods can includeintroducing a biocompatible ferrofluid, which may include mixing thebiological sample with the ferrofluid in the device as well as the useof the ferrofluid as a sheathing fluid flow in the operation of thedevice.

The methods are devices are capable of high throughput. In some aspects,the throughput is about 6 milliliters to about 25 milliliters of thebiological sample per hour. In some aspects, the flow rate is about theflow rate is about 10 μL to about 600 μL per minute.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1: iFCS Technology and Device (Medium Magnetic Field Gradient,One-Stage, Sheath-Free)

Circulating tumor cells (CTCs) contains abundant information regardingthe location, type and stage of cancer and have significant implicationsin both diagnostic target and guiding personalized treatment. Thetraditional isolation technologies rely on the properties of CTCs suchas antigens (e.g., epithelial cell adhesion molecule or EpCAM) or sizeto separate them from blood. Integrated—ferrohydrodynamic cellseparation (iFCS), a size-independent and marker-independent method, canisolate cancer cells with large size distribution in biocompatibleferrofluids with a throughput 6 mL h⁻¹, an average recovery rate of97.9%, and an 99.95% WBC depletion. We performed systematic parametricstudies of key factors influencing the performance of iFCS anddetermined parameters for high-throughput, high recovery rate and highpurity CTC separation. We then tested and validated the performance ofthe method with cancer cells from 8 cultured cancer cell lines and 3different types of cancer. The mean recovery rate of non-small cancercells from RBC-lysed blood using this technology was 98.7%. This methodwas also validated with small lung cancer cells and the mean recoveryrate was 95.5%.

Integrated Ferrohydrodynamic Cell Separation (iFCS) Working Principle

In the case of label-based magnetophoresis (FIG. 1.1A), magnetization ofthe particles {right arrow over (M)}_(p) is larger than the surroundingfluid medium {right arrow over (M)}_(f), therefore the magnetic forcewill point toward magnetic field maxima (FIG. 1.1A). On the other hand,for label-free negative magnetophoresis, magnetization of thediamagnetic particles {right arrow over (M)}_(P) is smaller than thesurrounding ferrofluid medium {right arrow over (M)}_(f), therefore themagnetic force will point toward magnetic field minima (FIG. 1.1B).Integrated ferrohydrodynamic cell separation (iFCS) scheme integratesboth label-based magnetophoresis and label-free negative magnetophoresisin a single device (FIGS. 1.1D and 1.1E) under non-uniform magneticfield. Particles and/or cells of magnetization larger than surroundingmedium and smaller than surrounding medium will move in differentdirection in the device, as shown in FIGS. 1.1C and 1.1F, resulting inseparation of particles and/or cells based on their relativemagnetization to the fluid medium.

Simulation and Calibration with Microbeads

In order to test the validity of working principle, we comparedsimulated trajectories of microbeads with experimental ones that wereobtained from imaging 15.0-μm-diameter non-magnetic beads (NMB) and11.8-μm-diameter magnetic beads (MB) in an iFCS device. From thetrajectories, we calculated the deflection in the y-direction, denotedas Y, and separation distance between the two types of beads, denoted asΔY. The simulation results were carried out using different parametersincluding throughput (10-600 μL min⁻¹), ferrofluid concentration(0-1.0%, v/v) and magnetic field gradient (20-280 T m⁻¹). The goal herewas to optimize the separation of non-magnetic beads from magneticbeads, which translated to maximizing both Y and ΔY simultaneously.Experimental conditions for calibration were the same as those insimulation. We extracted Y and ΔY at the end of the channel and usedthem to compare simulation and experimental results.

We first optimized the throughput of the device. Both simulation andexperimental results (FIG. 1.2A) showed a monotonically decreasing trendfor ΔY as the throughput increased. Both simulation (FIG. 1.2D) andexperimental results (FIG. 1.2G) indicated widely distributedtrajectories for 11.8 μm magnetic bead, and completely deflectedtrajectories for 15.0 μm non-magnetic beads at high flow rate. Thesecond parameter we optimized was ferrofluid concentration. In general,a higher ferrofluid concentration resulted large magnetic force onnon-magnetic beads, leading to a larger deflection in the y-direction.However, the Y value of magnetic beads (magnetic content is 0.737% v/vin this case) decreased as ferrofluid concentration increased, andincreased when the concentration was larger than 0.737% (FIGS. 1.2E and1.2H), resulting a smaller separation distance ΔY (FIG. 1.2B). It shouldbe noted that a portion of magnetic beads were trapped at the bottom ofchannel when ferrofluid concentration was low. The last parameter thatwe chose to optimize was magnetic field gradient, whose value changed aswe adjusted the distance between the magnet and the channel. FIGS. 1.2Cand 1.2F showed that, in both simulation and calibration, when themagnetic field gradient increased, the overall deflection Y for bothbeads increased too. This is because the magnetic force on beads wasdetermined in part by the gradient. The larger the field gradient, thelarger the magnetic force. On the other hand, the separation distancedecreased when the gradient was too weak to deflect beads. As a result,we chose a field gradient of 132 T m⁻¹ for following experiments.

To achieve high throughput, recovery rate and purity, we chose athroughput of 100 μL min⁻¹, ferrofluid concentration of 0.049% (v/v) andmagnetic field gradient of 132 T m⁻¹. With optimized parameters, we runa series of experiments with 20.3 and 8.0 μm (red fluorescent)non-magnetic beads and 11.8 μm (yellow fluorescent) magnetic beads. Allthe beads are randomly distributed near the inlet. Without magneticfield, there are no separation (FIG. 1.3D) and they start to deflectunder the non-uniform magnetic field (FIG. 1.3E). Non-magnetic beadswere collected from outlet 1, as shown in FIGS. 1.3C and 1.3E. on theother hand, magnetic beads were collected from outlet 2 and 3, as shownin FIG. 1.3F.

Verification of iFCS for High-Throughput and High-Recovery Spiked CancerCells Separation.

In order to show the size-independent characteristic of iFCS device, wemeasured the size of cancer cell lines before separation. The sizedistributions of various cancer cell lines and white blood cells arepresented in FIG. 1.4A. There is large size overlap between cancer celllines and white blood cells (WBCs), which was the main challenge inexisting label-free cancer cell separation methods. For example, theminimum size of PC-3 cell line was measured to be around 8 μm. Thismakes it challenging to enrich small PC-3 cancer cells from WBCs,because they have overlapping sizes (FIG. 1.4A).

In addition, we measured the number of dynabeads on each labeled WBC(n=1000). Human whole blood was obtained from healthy donors accordingto a protocol approved by Institutional Review Board (IRB) at Universityof Georgia. Calculated the amount of biotinylated antibody and magneticbeads required based on the WBC count. Used 100 fg/WBC for anti-CD45,anti-CD16 (BioLegend, San Diego, Calif.), and anti-CD66b (LifeTechnologies, Carlsbad, Calif.). Each WBC were labeled with 125 magneticbeads (Dynabeads Myone streptavidin T1, Life Technologies, Carlsbad,Calif.). Dynabeads were washed twice with 0.01% TWEEN 20 in PBS, thenwashed with 0.1% BSA in PBS and resuspended in PBS. The whole blood wasfirstly labeled with antibodies for 30 min and lysed by RBC lysis buffer(eBioscience, San Diego, Calif.) for 7 min at room temperature. Cellmixtures were centrifuged for 5 min at 800×g and the pellet weresuspended in PBS with Dynabeads. Incubate the tube for 25 min on therocker. Added ferrofluid and 0.1% (v/v) Pluronic F-68 non-ionicsurfactant (Thermo Fisher Scientific, Waltham, Mass.) to achieve thesame volume with whole blood and keep mixing for 5 more minutes. Thenumber of dynabeads on each WBC were counted with microscope and theaverage was 34±11 dynabeads per WBC (FIG. 1.4B). The magnetic content indynabeads was 11.5% (v/v). Based on the size of WBCs and the number oflabeled dynabeads, we calculated the magnetic content in each WBC (FIG.1.4C). About 99.5% of WBCs had magnetic content larger than 0.05% (v/v).Under optimized experimental conditions (throughput=100 μL min⁻¹,ferrofluid concentration=0.049%, and field gradient=132 T m⁻¹), wecollected about 99% spiked cancer cells and remove >99.5% WBCs.

We performed a series of experiments with spiked cancer cells ofcultured cell lines and WBCs based on optimal parameters. We firststudied the performance of iFCS using spiked PC-3 prostate cancers inWBCs. The concentration of DAPI stained WBCs, labeled with dynabeads,was 1×10⁶ cells mL⁻¹; CTCs were simulated by spiking ˜1×10⁴ CellTrackerGreen stained PC-3 cancer cells into 1 mL of WBCs. FIG. 1.5A indicatedthat all the cells were randomly distributed at the inlet of the device,then started to deflect when they entered the magnetic field region andwere completely separated at the outlets. PC-3 cells collected fromcollection outlet had polydisperse sizes, as shown in FIG. 1.5B. Tofuture validate the size-independent characteristics of iFCS, werepeated the experiments with MCF-7 and MDA-MB-231 cancer cells underthe same conditions. The size distributions of each cell line wereextremely similar before and after iFCS processing (FIG. 1.5C, Table1.1). WBCs collected from collection outlet were also identified bycounting the number of dynabeads on their surface (FIG. 1.5D). Theaverage number of dynabeads per WBC increased as the ferrofluidconcentration increased.

TABLE 1.1 Statistics about spiked and collected cancer cells underoptimized experimental conditions. Minimum Minimum cell average averagediameter diameter diameter diameter Cancer cell (spiked, (collected,(spiked, (collected, line μm) μm) μm) μm) MCF-7 7.0 6.6 17.5 ± 4.3 18.8± 5.3 MDA-MB-231 7.0 7.0 19.1 ± 3.6 19.9 ± 4.6 PC-3 8.1 6.6 19.9 ± 4.620.4 ± 4.9

We characterized the iFCS device with 6 types of cancer cells underoptimized conditions with ˜100 cell mL⁻¹ spike ratio. As shown in FIG.1.5E and Table 1.2, the average recovery rates of 97.9±1.0%, 97.6±1.0%,98.8±1.4%, 99.4±0.6%, 98.7±0.6%, and 99.7±0.6% were achieved for MCF-7,MDA-MB-231, HCC1806, H1299, H3122 and, PC-3 cell lines, respectively.The corresponding purities of separated cancer cells for each cell linewere 22.0±1.3% (MCF-7), 23.5±0.7% (MDA-MB-231), 25.2±1.5% (HCC1806),23.1±0.9% (H1299), 22.2±0.9 (H3122), and 23.3±0.4 (PC-3), confirming therobustness of the iFCS device for cancer cell separation. We furthervalidated the device with small lung cancer cells. The average recoveryrates of 95.0±1.2% and 95.9±1.3% were achieved for DMS79 and H69 celllines, respectively. A series of spike-in experiment, in which a certainnumber PC-3 cells (100, 250, 500, 1000, 2000) and 1 million WBCs werespiked into 1 mL ferrofluid, were carried out to validate that thedevice has the potential to process clinically relevant blood samples(FIG. 1.5F). An average recovery rate of 98.8% was achieved in the iFCSfor this particular prostate cancer cell line.

TABLE 1.2 Cancer cell separation under optimized conditions. ~100 cancercells and 1 × 10⁶ WBCs were spiked into 1 mL of ferrofluid withconcentration of 0.049% (v/v). The recovery rate was defined as theratio of the number of cancer cells collected from outlet 1 over thetotal number of spiked cancer cells from all outlets. The purity wasdefined as the number of identified cancer cells over the total numberof cells (WBCs + cancer cells) from outlet 1. Data are expressed as mean± standard deviation (s.d.), n = 3. Measured Measured No. of No. ofminimum Average spiked cancer No. of Cancer Cancer diameter diametercancer cells cells cell line cell type (μm) (μm) cells (outlet 1)(outlet 2 & 3) Recovery rate No. of WBCs Purity MCF-7 Breast 7.0 17.5110 ± 9 108 ± 8 2 ± 1 97.9 ± 1.0 384 ± 2 22.0 ± 1.3 MDA-MB-231 Breast7.0 19.1 110 ± 7 108 ± 7 3 ± 1 97.6 ± 1.0 354 ± 6 23.5 ± 0.7 HCC1806Breast 6.7 15.4 109 ± 4 108 ± 5 1 ± 2 98.8 ± 1.4 324 ± 6 25.2 ± 1.5H1299 Lung 7.2 20.4 101 ± 4 100 ± 3 1 ± 1 99.4 ± 0.6 336 ± 4 23.1 ± 0.9H3122 Lung 6.7 17.7 104 ± 3 103 ± 3 1 ± 1 98.7 ± 0.6 360 ± 2 22.2 ± 0.9DMS79 Lung 3.2 12.8  118 ± 12  112 ± 10 6 ± 2 95.0 ± 1.2 350 ± 4 24.2 ±1.7 H69 Lung 4.0 11.2 106 ± 4 102 ± 4 4 ± 1 95.9 ± 1.3 366 ± 7 21.7 ±0.8 PC-3 Prostate 8.1 19.4  99 ± 3  99 ± 3 0 ± 1 99.7 ± 0.6 324 ± 1 23.3± 0.4

Example 2: iFCS Technology and Device (High Magnetic Field Gradient,Multiple-Stage, Sheath-Flow)

In this device, we have developed a marker-independent andsize-independent integrated ferrohydrodynamic cell separation (iFCS)method, which is biocompatible and could enrich entire rare circulatingtumor cells (CTCs) from patient blood with a high throughput and a highrecovery rate. The blood samples suspended in ferrofluids (0.03% v/v)are injected into the inlet A and focused by sheath flow through inlet B(ferrofluids with a concentration of 0.03% v/v). The majority of labeledwhite blood cells (WBCs) and free Dynabeads are depleted into the wasteoutlet 1 in stage 2. The unlabeled cells and some labeled WBCs,including CTCs, enter the stage 3. The CTCs are then continuouslydeflected to the two sides of the channel and labeled WBCs and freebeads are focused in the center of the channel, as shown in FIGS.1.6A-1.6C.

The magnet holder consists of top and bottom parts and is secured withscrews and nuts. Two magnet arrays repelling each other are placed inthe top and bottom holders, respectively. The microfluidic device issandwiched between the magnet holders and the center of the stage 3'schannel is exactly aligned with the center of the magnet arrays (FIGS.1.6A-1.6C and FIG. 1.7). CTCs enrichment is achieved by inputting redblood cell (RBC)-lysed blood that is pre-labeled with magnetic beads(Dynabeads MyOne Streptavidin T1) targeting WBCs via CD45, CD16 andCD66B surface antigens (FIG. 1.8). We will refer this multiple stagedevice as FCSv2.

We performed systematic optimization of this method and determinedparameters in the microfluidic device that achieved an average recoveryrate of 99.16% using 8 different cell lines (HCC1806, HCC70, MCF7,MDA-MB-231, H1299, H3122, DMS79, and H69) from RBC-lysed WBCs, whichwere labeled with Dynabeads conjugated with leukocyte antibodies. Thedeveloped device is able to process 12 mL of blood within one hour. Thedevice achieved an average WBC carryover of 527 WBCs per mL of inputblood. Specifically, for each cell line at ˜100 cells per mL spikeratio, the recovery rates of cancer cells were 98.46±0.50% (HCC1806breast cancer), 99.68±0.46% (HCC70 breast cancer), 99.05±0.75% (MCF7breast cancer), 99.35±0.46% (MDA-MB-231 breast cancer), 99.40±0.85%(H1299 non-small cell lung cancer), 99.13±0.49% (H3122 non-small celllung cancer), 99.11±1.25% (DMS79 small cell lung cancer), and99.11±0.74% (H69 small cell lung cancer). To validate that the devicehas the potential to process clinically relevant blood samples, a seriesof spike-in experiments in which a certain number of HCC70 breast cancercells (20, 50, 100, and 200) are spiked into 1 mL of WBCs. As shown inFIGS. 1.9A-1.9B, an average recovery rate of 99.08% is achieved in theFCSv2 device. FIGS. 1.10A-1.10B show the separation of spiked cancercells (stained with CellTrack Green) at the end of stages 2 and 3.

Example 3: Optimized iFCS System

Introduction

Isolation of circulating tumor cells (CTCs) from blood provides aminimally-invasive alternative into the basic understanding, diagnosisand prognosis of metastatic cancers. The roles and clinical values ofCTCs are under intensive investigation, yet most studies are limited bytechnical challenges in the comprehensive enrichment of intact andviable CTCs with minimal white blood cells contamination. The presentexample describes a novel, optimized system and method based on contrastof cell magnetization, termed as integrated ferrohydrodynamic cellseparation (iFCS), that enriches CTCs in a tumor antigen-independent andcell size variation-inclusive manner, and achieves high-throughput (12mL h⁻¹), high recovery rate (99.16% at down to ˜10 cells mL⁻¹ spikeratio), low WBC contamination (˜500 cells for every one milliliter bloodprocessed) and is biocompatible. This method allows for studies onsubtypes of CTCs, and their correlations with clinical and diagnosticvariables in small cohorts of cancer patients.

Insights on heterogeneity among circulating tumor cells (CTCs) havesignificant implications for basic and translational research ofmetastatic cancer that is responsible for over 90% of cancer relatedmortality. 1-4 While primary tumor characterization is the most commonsource of material to predict tumorigenesis, clinically relevantfindings would include the ability to predict whether the tumor willlikely metastasize and establish lethal colonies of tumors in distalorgan sites. Due to inherent heterogeneous composition of primarytumors, needle-biopsies and surgical samples may miss key diagnosticmarkers that would define metastatic potential of the tumor.Characterizing blood borne circulating tumor cells provides a windowinto metastasis research as tumor cells are in route to their new niche,where these cells represent disease potential of the tumors to establishmultiple sites. 3⋅4 Hence, CTCs could be a more representative sample oftumor disease potential than a primary tumor biopsy, including acompendium of genetic changes that increase metastatic potential overthe course of tumor evolution. Development of innovative technologiesthat will allow the enrichment and characterization of a completerepertoire of viable CTCs could increase our understanding of metastasisand may lead to novel applications including the creation of in vitroand ex vivo models to experimentally manipulate and screen panels ofpatient derived tumors.

Three concurrent technical challenges in existing CTC enrichmentmethods, including the dependence of specific tumor antigens for tumorcell recognition, inability to account for the variation of tumor cellsizes in isolation, and difficulty of keeping CTCs viable and intact fordownstream analysis, complicated the study and applications of CTCs.These issues are worsened by the fact that CTCs are extremely rare,estimated at less than 10 tumor cells in every one-milliliter of wholeblood. Past studies have showed that CTCs isolated by the US Food andDrug Administration (FDA) approved CellSearch system, identified byepithelial cell adhesion molecules (EpCAM) alone, were associated withpoor prognosis in metastatic and localized carcinomas in clinicaltrials.5-7 However, increasingly CTCs were found to be a rare andheterogeneous population of different phenotypic subtypes,1⋅8 in which afraction of original epithelial tumor cells could transition intostem-like mesenchymal cells in a metamorphosis noted as EMT, Epithelialto Mesenchymal Transition.3 This transition may be what gives CTCs thetraits of high motility, invasiveness and limitless potential to createa new tumor site, therefore cells that have gone through this transitioncould possess the greatest threat of metastasis and short-termrecurrence.3⋅4⋅9 Given the importance of EMT CTCs, the influence ofmesenchymal properties on the prolonged survival of CTCs in thecirculation, and on their capacity to form metastatic tumors, newmethods are urgently needed to allow for a comprehensive enrichment andanalysis of viable CTCs. Microfluidics-based methods have provided a newavenue to enrich and study CTCs for the past decade but were oftenbiased because of the use of specific tumor antigens or cell sizethreshold in enrichment. Majority of microfluidic methods operated basedon either marker-dependent or cell size-dependent principles.10 Forexample, marker-dependent methods that relied on EpCAM or othercombination of tumor cell surface antigens were rendered ineffective dueto inherent heterogeneity of tumor subtypes.11 The significantdifference among various markers and their expression levels in CTCsundergoing EMT was difficult to predict, resulting in incompleterecovery of CTCs from clinical samples. On the other hand, cellsize-dependent methods including those based on filtration,12 dean flowand vortex chip,13⋅14 depleted blood cells and recovered CTCs that werelarger than ˜10 μm in diameter, based on a presumed size differencebetween blood and cancer cells.10 The issue of these methods was that asignificant percentage of CTCs in circulation were comparable or smallerthan blood cells. Our measurements of white blood cells (WBCs) andcultured cancer cells revealed that there was a significant size overlapbetween the two, and an appreciable percentage (e.g., ˜35% for DMS 79and H69 small cell lung cancer cell lines) of cancer cells were smallerthan ˜10 μm. In addition, clinically isolated CTCs were reported to beas small as ˜6 μm.15 As a result, few cell size-dependent methods couldachieve complete recovery and low WBC contamination simultaneously.10⋅16Furthermore, CTCs are fragile and need to be processed with gentleenrichment conditions to keep their viability and tumorigenic capabilityfor downstream studies. In summary, the inherent bias in tumorantigen-dependent and cell size-dependent methods, and the recognitionthat CTCs are highly rare, heterogeneous and fragile, highlight the needto develop new methods that can enrich viable CTCs regardless of theirsurface antigen and size profiles.

The device/system developed and described in the present Exampleaddressed the above-mentioned challenges through the development of anovel CTC enrichment method that is based on contrast of magneticproperties of cells, termed as integrated ferrohydrodynamic cellseparation (iFCS). This method is tumor antigen-independent and cellsize variation-inclusive; it allows for simultaneous depletion of WBCsand enrichment of viable CTCs, resulting in complete recovery of intactand viable CTCs with minimal WBC contamination that were suitable forclinical applications. In developing this method, we performedsystematic parametric studies of key factors influencing the performanceof iFCS and determined parameters for high-throughput (12 mL h⁻¹), highrecovery rate (99.16% at down to ˜10 cells mL⁻¹ spike ratio), low WBCcontamination (˜500 cells for every one milliliter blood processed) andbiocompatible enrichment (cell viability of 97.69±0.70% afterenrichment) of CTCs from the blood of cancer patients. iFCS was firstvalidated with cancer cells from 8 cultured cell lines of 3 differenttypes of cancer. Mean recovery rate of cancer cells from red blood cell(RBC)-lysed blood using this method was 99.16%. The prototype iFCSdevice carried over on average 533±34 WBCs per 1 mL of blood processed.Enriched cancer cells had excellent short-term viability, and intactcapability to proliferate to confluence. Clinically, iFCS was first usedto study cell size variation and surface antigen expressionheterogeneity of CTCs enriched from 3 breast cancer patients and 3 lungcancer patients. This study revealed a high degree of variation in CTCsizes. 55.4% of patient derived CTCs possessed an effective diameter ofless than 10 μm, and there was a significant overlap in sizes betweenCTCs and WBCs. The study also showed heterogeneity of epithelial andmesenchymal characteristics in patient CTCs' surface antigen expression.These results highlighted the need for tumor antigen-independent andcell size variation-inclusive methods such as iFCS. We also used iFCS ata remote site (Henry Ford Health System, Detroit, Mich.) to investigatewhether variable counts of CTC subtypes would correlate with clinicaland diagnostic variables. We found, within a small cohort (n=6) of earlystage breast cancer patients, that mesenchymal and EMT subtypes of CTCshad a higher correlation to tumor grade than epithelial subtype.

Methods

Model of iFCS and its validation. We developed an analytical model usedin this study to simulate cell trajectories in three-dimensional (3D)manner.18, 19 It could predict 3D transport of diamagnetic cancer cellsand magnetic WBCs in ferrofluids inside a microfluidic channel coupledwith permanent magnets. The magnets produced a spatially non-uniformmagnetic field that led to a magnetic force on the cells. Trajectoriesof the cells in the device were obtained by (1) calculating the 3Dmagnetic force via an experimentally verified and analyticaldistribution of magnetic fields as well as their gradients, togetherwith a nonlinear Langevin magnetization model of the ferrofluid and (2)solving governing equations of motion using analytical expressions ofmagnetic force and hydrodynamic viscous drag force in MATLAB (Math WorksInc., Natick, Mass.).

Synthesis and characterization of ferrofluids. Maghemite nanoparticles(10.91±4.86 nm) were synthesized by a chemical co-precipitation methodas previously described.19 Size and morphology of maghemitenanoparticles were characterized via transmission electron microscopy(TEM; FEI, Eindhoven, the Netherlands). Magnetic properties of theferrofluid were measured at room temperature using a vibrating samplemagnetometer (VSM; MicroSense, Lowell, Mass.). The viscosity offerrofluids was characterized with a compact rheometer (Anton Paar,Ashland, Va.) at room temperature. Ferrofluid characterization data areillustrated in FIGS. 3.7A-3.7C.

Cell culture and sample preparation. Cancer cell lines (ATCC, Manassas,Va.) including four breast cancer cell lines (HCC1806, HCC70, MCF7 andMDA-MB-231), two NSCLC cell lines (H1299 and H3122), two SCLC cell lines(DMS79 and H69), and one prostate cancer cell line (PC-3) were used inthis study. Cell lines were cultured following manufacturer'srecommended protocol. Cancer cells were fluorescently stained withCellTracker Green (Life Technologies, Carlsbad, Calif.) before each use.Cells were counted with a Nageotte counting chamber (Hausser Scientific,Horsham, Pa.) to determine the exact number of cells per μL. Desiredcancer cells (10, 25, 50, 100 or 200) were spiked into 1 mL of labeledWBCs.

Ferrofluid biocompatibility. Short-term cell viability after iFCS wasexamined using a Live/Dead assay (Life Technologies, Carlsbad, Calif.).For long-term proliferation, separated HCC1806 cells from an iFCS devicewere washed three times with culture medium to remove the nanoparticles,and then the cells were suspended in culture medium and seeded into aT25 flask. Cells were then cultured at 37° C. under a humidifiedatmosphere of 5% CO2. Cellular morphology was inspected every 24 hours.

iFCS device fabrication and assembly. Microfluidic devices were made ofpolydimethylsiloxane (PDMS) using standard soft lithography techniques.The thickness of the microfluidic channel was measured to be 300 μm by aprofilometer (Veeco Instruments, Chadds Ford, Pa.). Device was placedwithin a custom aluminum manifold that held four NdFeB permanent magnets(K&J Magnetics, Pipersville, Pa.) in a quadrupole configuration. Eachmagnet was 50.8 mm in length, 6.35 mm in both width and thickness, andhad a remanent magnetization of 1.48 T.

Microfluidic experiment setup and procedure. During a typicalexperiment, a microfluidic device inserted within manifold was placed onthe stage of an inverted microscope (Axio Observer, Carl Zeiss, Germany)for observation and recording. Two fluid inputs were controlled byindividual syringe pumps (Chemyx, Stafford, Tex.). Blood samples wereinjected into an inlet of an iFCS device, sheath flow of ferrofluids wasinjected into a second inlet. Images and videos of cells were recordedwith a CCD camera (Carl Zeiss, Germany). After enrichment, cells werecollected in a 15-mL centrifuge tube with complete culture media.Fabricated devices were flushed by 70% ethanol for 10 minutes and thenprimed with 1× phosphate-buffered saline (PBS) supplemented with 0.5%(w/v) bovine serum albumin (BSA) and 2 mM EDTA (Thermo FisherScientific, Waltham, Mass.) for 10 minutes before each use.

Patient recruitment and sample processing. Healthy human blood sampleswere obtained from Clinical and Translational Research Unit ofUniversity of Georgia with informed consents according to a protocolapproved by Institutional Review Board (IRB) (University of Georgia,STUDY00005431). Healthy donor samples were used for spike-in experimentswith cell lines. Cancer patient samples collected at University Cancerand Blood Center (Athens, Ga.) was approved by the IRB (University ofGeorgia, STUDY00005431) before study initiation and informed consent wasobtained from the participants. Cancer patient samples collected atHenry Ford Health System (Detroit, Mich.) was approved by the IRB (HenryFord Health System, Davis-11564) before study initiation and informedconsent was obtained from the participants. All blood samples werecollected into either vacutainer K2-EDTA tubes (BD, Franklin Lakes,N.J.) or cell-free DNA BCT (Streck, Omaha, Nebr.) and were processedwithin 2 hours of blood draw. Detailed patient information and CTCenumeration was listed in Table 2.5. Complete blood count (CBC) reportswere obtained to determine the number of WBCs. Whole blood was labeledwith leukocyte-specific biotinylated antibodies including anti-CD45(eBioscience, San Diego, Calif.), anti-CD16 (eBioscience, San Diego,Calif.), and anti-CD66b (BioLegend, San Diego, Calif.) for 30 min. Theantibody conjugated blood was lysed by RBC lysis buffer (eBioscience,San Diego, Calif.) for 10 minutes at room temperature. Blood cells werethen incubated with streptavidin coated magnetic Dynabeads (LifeTechnologies, Carlsbad, Calif.) for 30 minutes on a rocker. All thelabeling and washing procedures were followed by manufacturer'srecommended protocol. Blood cells were finally suspended in the samevolume of ferrofluid (0.028% v/v) containing 0.1% (v/v) Pluronic F-68non-ionic surfactant (Thermo Fisher Scientific, Waltham, Mass.) beforeprocessing.

CTC identification. After processing of blood with an iFCS device, cellswere immobilized onto a poly-1-lysine coated glass slide with acustomized cell collection chamber. The collected cells were fixed with4% (w/v) paraformaldehyde for 10 minutes and subsequently permeabilizedwith 0.1% (v/v) Triton X-100 in PBS for 10 minutes. Cells were thenblocked with blocking reagent (Santa Cruz Biotechnology, Dallas, Tex.)for 30 minutes. After blocking nonspecific binding sites, cells wereimmunostained with primary antibodies including anti-cytokeratin(CK3-6H5)-FITC (Miltenyi Biotec, Auburn, Calif.) or EpCAM (EBA-1)-AlexaFluor 488 (Santa Cruz Biotechnology, Dallas, Tex.), Vimentin (V9)-AlexaFluor 647 (Santa Cruz Biotechnology, Dallas, Tex.), and N-cadherin(13A9)-Alexa Fluor 594. Nuclei were counterstained with DAPI. Allsamples were also stained with anti-CD45 (H130)-PE (BD Biosciences, SanJose, Calif.) to identify leukocytes. After immunofluorescence staining,cells were washed with PBS and coverslipped with mounting medium forimaging or stored at 4° C.

CTC culture. Primary cells from patient blood after processing with iFCSdevice were centrifuged at 200×g for 5 minutes at 37° C. and resuspendedin RPMI-1640 media with 15% fetal bovine serum (FBS). Cells werecultured in vented T-25 cm2 flask at 37° C. with 5% CO2. Primary cellgrowth rate was determined over a 72-hour period.

Results

Overview of iFCS.

Integrated ferrohydrodynamic cell separation (iFCS) method uses thefollowing strategy to achieve tumor antigen-independent and cell sizevariation-inclusive enrichment of viable CTCs and simultaneous depletionof contaminating WBCs, leaving intact CTCs at its device's output withminimal WBC carryover. In this strategy, virtually all WBCs are renderedmagnetic by labeling them with magnetic microbeads through a combinationof leukocyte biomarkers, while CTCs remain unlabeled. WBC-beadconjugates and CTCs continuously flow through a microfluidic devicefilled with ferrofluids, a colloidal suspension of magneticnanoparticles with tunable particle concentration. Magnetization of theferrofluid M_(fluid), under an external magnetic field, is adjusted tobe less than that of WBC-bead conjugates M_(WBC-bead), so that unlabeledCTCs with a close to zero magnetization M_(CTC), regardless of theirsizes, are pushed towards a magnetic field minima due to a phenomenonknown as “diamagnetophoresis” (FIG. 2.1A, top),17 while WBC-beadconjugates are attracted to a magnetic field maxima through acompetition between both “magnetophoresis” and “diamagnetophoresis”(FIG. 2.1A, bottom), and continuously depleted. The strategy integratesboth “diamagnetophoresis” and “magnetophoresis” in ferrofluids to enrichthe entire repertoire of CTCs from blood regardless of CTCs' surfaceantigen profile and size profile.

The iFCS-based CTC enrichment strategy relies on the establishment ofboth “magnetophoresis” and “diamagnetophoresis” of cells immersed inferrofluids. A magnetic force is generated on magnetic or diamagneticcells under a non-uniform magnetic field,17{right arrow over (F)} _(mag)=μ₀ V _(cell)[({right arrow over (M)}_(cell) −{right arrow over (M)} _(fluid))⋅∇]{right arrow over (H)}  (1)

where μ₀=4π×10⁻⁷ H m⁻¹ is the permeability of free space, V_(cell) isthe volume of the cell, {right arrow over (M)}_(cell) is itsmagnetization, {right arrow over (M)}_(fluid) is magnetization of theferrofluid surrounding the cell, and {right arrow over (H)} is magneticfield strength at the center of the cell. For cells in ferrofluids undera magnetic field, magnitudes of the magnetization of the cell M_(cell)and the ferrofluid M_(fluid) with superparamagnetic particles in it canbe modeled via a Langevin function. From Eq. 1, we learn that themagnetic force directs cells to either a magnetic field maxima or minimadepending on the contrast between cell and fluid magnetizations, i.e.,the sign of the term {right arrow over (M)}_(cell)−{right arrow over(M)}_(fluid), and the magnitude of the force is also proportional to thevolume of the cells V_(cell).

FIGS. 2.1A-2.1D illustrate the design of a prototype iFCS microfluidicdevice based on the above principle. We incorporated two enrichmentstages in prototype devices, in order to prevent magnetic microbeadaggregation due to the use of large number of magnetic beads. Prior todevice processing, WBCs in blood were labeled with magnetic microbeadsthrough leukocyte surface biomarkers so that overall magnetization ofthe WBC-bead conjugates M_(WBC-bead) was larger than its surroundingferrofluid medium M_(fluid). Magnetization of the unlabeled CTCs M_(CTC)was close to zero and less than its surrounding ferrofluids M_(fluid).In the first stage (FIG. 2.1B, top), a magnetic field was used to directunlabeled and sheath-focused CTCs to remain at the upper boundary of amicrochannel, while attract unbound magnetic beads and WBCs labeled with≥3 microbeads towards a waste outlet. This way, a significant percentageof beads and WBCs were depleted before the second stage, so that beadaggregation was minimized (FIG. 3.1A). In the second stage, a symmetricmagnetic field with its maximum at the middle of the channel was used toattract remaining WBC-bead conjugates towards to the channel center forfast depletion, while unlabeled CTCs flowing along the upper and lowerchannel boundaries were collected for analysis at the end of the channel(FIG. 3.1B). This design aimed to enrich all CTCs regardless of theirsurface antigens and sizes, at the same time remove virtually all WBCsfrom collection outlets. An example of an arrangement of magneticsources (in this case, a magnetic array) relative to the first andsecond separation stages of an exemplary device is illustrated in FIG.3.8.

A physical model was developed to optimize the prototype device forpractical CTC enrichment. CTCs are extremely rare in the bloodcirculation and hidden among millions of WBCs with similar size. Therate of CTC occurrence was reported to be <10 cells in one milliliter ofblood.10⋅16 In order to optimize iFCS method and objectively evaluateits performance, four metrics were used, including cell-processingthroughput, CTC recovery rate, WBC contamination and integrity ofenriched cells, which were consistent with reports of existing methods(see Table 2.1). For iFCS, the parameters that affected these fourmetrics include device geometry, magnetic field and its gradient,magnetic bead labeling efficiency of WBCs, flow rate of cells, andferrofluid properties. These parameters were coupled and optimizedsystematically. We created such a model that could predictthree-dimensional (3D) trajectories of cells in laminar flow conditionsinside the device.18⋅19 Both magnetic force and hydrodynamic drag forcewere taken into consideration in simulating the cell trajectories.

Optimization of iFCS for CTC Enrichment.

We optimized iFCS for tumor antigen-independent and cell sizevariation-inclusive enrichment of CTCs, with a goal of enriching theentire repertoire of viable CTCs with minimal WBC contamination. Inquantitative terms, the performance goals for iFCS devices included: (1)a complete CTC recovery rate of >99% at clinically relevant occurrencerate for CTCs (1-10 cells mL⁻¹), regardless of their surface antigensand sizes; (2) a minimal WBC contamination of ˜500 cells at the deviceoutput for every one milliliter blood processed, (3) a blood processingthroughput of more than 10 mL h⁻¹, and (4) unaffected cell integrityafter enrichment, including viability and proliferation. These metricswere chosen as targets after a survey of existing microfluidic CTCenrichment methods (see Table 2.1).

Systematic optimization of iFCS devices focused on the effects of devicegeometry, magnetic microbeads functionalized per WBC, magnetic field andits gradient, flow rates, as well as ferrofluid concentration on deviceperformance, including throughput, recovery rate and WBC contamination.Firstly, we determined microchannel dimensions for both stages bybalancing a clinical goal of processing at least 10 milliliters of bloodwithin one hour, and a goal to maintain laminar flow in the device.Final channel dimensions (first stage: 55×1.6×0.3 mm; second stage:55×1.2×0.3 mm; L×W×H) were optimized so that the Reynold's number was onthe order of 10 when cell flow rate was 12 mL h⁻¹, ensuring laminar flowcondition and physiologically equivalent shear rates (first stageaverage: 270.8 s⁻¹, range: 63.4-510.4 s⁻¹; second stage average: 190.8s⁻¹; range: 54.4-360.5 s⁻¹) during CTC enrichment.20 The prototypemicrochannel and assembled device are shown in FIGS. 2.1C and 2.1D.Secondly, the amplitude of magnetic force on cells is proportional tothe amplitude of magnetic field gradient. In order to maximize fieldgradient, we adopted a quadrupole magnet configuration in the iFCSdevice that could generate magnetic flux density in the range of 0.5-1.5T, and magnetic flux density gradient up to 625 T m⁻¹ (FIG. 2.2A).Thirdly, number of magnetic microbeads functionalized onto WBCs shouldbe maximized to increase the contrast between WBC-bead conjugates andsurrounding ferrofluids. Therefore, we optimized a WBC functionalizationprotocol by using a combination of three leukocyte surface biomarkers.Streptavidin-coated Dynabeads (1.05 μm, 11.4% Fe₂O₃ volume fraction) andbiotinylated anti-human CD45, CD15 and CD66b antibodies combination wereused. Results in FIG. 2.2B show that with antibody and beadconcentrations (CD45: 100 fg/WBC, CD15: 75 fg/WBC, CD66b: 75 fg/WBC,magnetic beads: 125/WBC), WBCs were conjugated with 34±11 beads,and >99.9% of WBCs were labeled. Average magnetic content volumefraction of WBCs from bead conjugation was 0.36%, with a minimal valueof 0.026%, corresponding to WBCs that were labeled with just onemagnetic bead. By choosing a ferrofluid concentration that was in thevicinity of the minimal value of WBCs' magnetic content volume fraction,it became possible to deplete virtually all WBCs.

The remaining optimization focused on the effect of ferrofluidconcentration and cell-processing throughput on the performance metricsin the second stage. For this part of optimization, we calculated twooutputs—a deflection in the y-direction for cells (see FIG. 2.2A forcoordinates), denoted as Y, and a separation distance between WBCs andCTCs, denoted as ΔY. Both outputs were optimized using parametersincluding ferrofluid concentration (0-0.04% v/v) and throughput (0-700μL min⁻¹, i.e., 0-42 mL h⁻¹). The goal was to maximize CTC recovery rateand minimize WBC contamination, which translated to maximizing ΔY andcell-processing throughput simultaneously. FIG. 2.2C shows thatseparation distance ΔY reached a maximum when using a ferrofluid with0.028% magnetic volume fraction, and largest throughput that could beachieved without compromising CTC recovery was 200 μL min⁻¹ (i.e., 12 mLh⁻¹).

In summary, the optimization resulted in following operating parametersfor the prototype iFCS devices: magnetic flux density in the range of0.5-1.5 T, and magnetic flux density gradient up to 625 T m⁻¹ viaassembling four NdFeB permanent magnets in quadrupole configuration;WBCs conjugated with 34±11 magnetic beads, and over 99.9% WBCs labeled;cell-processing throughput 12 mL h⁻¹; and ferrofluid concentration0.028% (v/v). Microchannels in the device had a thickness of 300 μm anda total length of 55 mm, widths of the microchannels for the first andsecond stages were 1600 μm and 1200 μm, respectively. Using theseparameters, we studied via simulation the recovery rate of CTCs (CTCsize range: 3-32 μm in spherical diameter) spiked into WBCs (WBC sizerange: 5-25 μm in spherical diameter, 34±11 magnetic beads per cell). Wechose the smallest diameter of CTCs to be 3 μm, a value that was almosthalf the size of smallest reported CTCs from clinical samples,15 inorder to test the robustness of the tumor antigen-independent and cellsize variation-inclusive enrichment method. FIG. 2.2D shows adistribution of cell locations at the end of each stage. In quantitativeterms, FIG. 2.2E reports that 96.35% of initial WBCs that were labeledwith ≥3 beads and all unbound beads were depleted after just the firststage while all CTCs were persevered, including the smallest 3 μm ones.After the second stage, 3.60% of initial WBCs that were labeled with 2-3beads were further removed without affecting CTCs. Overall, these twostages together were predicated to be able to deplete 99.95% of WBCs(corresponding to ˜500 WBCs contamination or carryover per 1 milliliterblood processed) and preserve 100% of CTCs regardless of their sizeprofile.

Validation of iFCS with Spiked Cancer Cells in Human Blood.

Using optimized device geometry and operating parameters, we studiedcancer cell enrichment in iFCS prototype devices using a total of 8cultured cancer cell lines that have drastically different average cellsizes and polydispersity, including 4 breast cancer cell (BrC) lines, 2non-small cell lung cancer cell (NSCLC) lines, and 2 small cell lungcancer cell (SCLC) lines. Performance of the enrichment was evaluated oncell-processing throughput, cell recovery rate, WBCcontamination/carryover, and integrity of isolated cells. These resultswere also compared to simulation for test the robustness of theanalytical model. A typical enrichment process can be visualized inFIGS. 2.3A and 2.3B, in which ˜100 green fluorescently stained HCC1806breast cancer cells (cell size range 6-47 μm) were spiked into 1 mL ofWBCs and processed in an iFCS device at a flow rate of 12 mL h⁻¹. In thefirst stage (FIG. 2.3A, top: phase contrast; bottom: epifluorescence),magnetic force attracted labeled WBCs and unbound beads toward a wasteoutlet while unlabeled cells including all CTCs and approximately 3.65%of WBCs were continuously flown to the second stage. No aggregation ofmagnetic beads or ferrofluids was observed within one hour of operation.In the second stage (FIG. 2.3B, top: phase contrast; bottom:epifluorescence), magnetic forces deflected unlabeled cancer cells fromthe mixture toward both upper and lower collection outlets. Meanwhile,labeled WBCs were focused to the middle channel and depleted through awaste outlet.

We continued by testing recovery rate and WBC carryover at cancer celloccurrence rates that were clinically relevant. The average rate ofrecovery for HCC70 breast cancer cells was 99.18% at spike ratiosbetween 10-200 (10, 25, 50, 100, and 200) cells mL⁻¹ and showed minimalvariations among three repeats (FIG. 2.3C), which was consistent withsimulation results. After cell enrichment, the device carried over onaverage 533±34 WBCs per 1 milliliter of blood processed. Much of thecarryover was derived from WBCs that were either not labeled or labeledwith just one magnetic bead (FIG. 3.2), which was predicted bysimulation results. The level of WBC contamination found in iFCS deviceswas comparable to the monolithic version of the CTC-iChip (˜445 WBCs per1 mL of blood processed),21 and lower than other recently reportedmethods, including magnetic ranking cytometry (2000 WBCs mL⁻¹) andprevious versions of CTC-iChip (1200 WBCs mL⁻¹).8⋅22⋅23 Aftersuccessfully demonstrating low-concentration cancer cell enrichmentusing HCC70 breast cancer (BrC) cell line, we expanded thecharacterization of recovery rate in iFCS devices with 7 other types ofcancer cell lines, including two SCLC cell lines. A measurement on thecell size of these cells lines and WBCs showed that there was asignificant size overlap between WBCs and cancer cells, especially theSCLC lines (FIG. 2.3D and Table 2.2). A noticeable percentage of patientCTCs were smaller than 10 μm (55.4%; FIG. 3.3). This would make theenrichment of cancer cells via size-dependent methods ineffective.However, as shown in FIG. 2.3E, by using iFCS method, recovery rates of98.46±0.50%, 99.05±0.75%, 99.35±0.46%, 99.40±0.85%, 99.13±0.49%,99.11±1.25%, and 99.11±0.74% were obtained for HCC1806 (BrC), MCF7(BrC), MDA-MB-231 (BrC), H1299 (NSCLC), H3122 (NSCLC), DMS79 (SCLC), andH69 (SCLC) cell lines, respectively. The average recovery rate across 8cancer cell lines was 99.16%, which indicated a complete recovery ofspiked cancer cells, including even the SCLC cells, regardless of theirsize profiles. The size distribution of three cells lines before andafter enrichment in a single stage version of the iFCS device furtherconfirmed that iFCS could enrich all cancer cells without a loss ofsmall ones (FIG. 2.3F and Table 2.3).

Ferrofluids and the iFCS enrichment process had little impact on cellviability and intactness, given the extremely low ferrofluidconcentration (0.028% v/v) and low shear rate in enrichment. We examinedshort-term cell viability and long-term cell proliferation of cancercells following the enrichment process. As shown in FIG. 2.3G, cellviability of HCC1806 breast cancer cells before and after enrichmentwere determined to be 98.30±0.56% and 97.69±0.70%, respectively,indicating a negligible decrease in cell viability before and after theiFCS process. Representative fluorescence images of cells are shown inFIG. 2.3H. We also studied whether enriched cancer cells continued toproliferate normally. FIG. 2.3I shows the images of enriched HCC1806breast cancer cells on the third day. They were able to proliferate toconfluence and maintain the morphology after the iFCS process.Fluorescence image in FIG. 2.3I also confirmed that cells were viableafter the 3-day culture. Enriched cells were intact and suitable forimmunofluorescent and cytopathological staining (FIG. 2.3J and FIG.3.4).

Profiling Cell Size and Surface Antigen Heterogeneity Among CTCs inCancer Patients

iFCS devices were capable of enriching CTCs regardless of their cellsize variation and surface antigen heterogeneity from clinical samplesof breast cancer patients. We investigated whether the heterogeneouspopulation of CTC cell types enriched from iFCS could potentially yieldgreater clinical utility. For this purpose, we studied two cohorts ofcancer patients (breast cancer and lung cancer). We quantified thenumbers and sizes of CTCs overall, then defined and quantified CTCsubtypes in each patient and found distinct quantities of CTC subtypeswithin the patient cohort. We categorized the CTC subtypes based ontheir expression of cell surface markers for epithelial and mesenchymalcell types.

In the first cohort, we used iFCS devices to process blood samples from3 breast cancer patients who were recruited and consented fromUniversity Cancer and Blood Center (Athens, Ga.) under an approved IRBprotocol (University of Georgia, STUDY00005431). These patients areidentified as breast cancer optimization cohort (BrC-P#-Opt, in which #indicates the number of patient) in this paper. Peripheral blood wascollected from the patients before initiation of treatment. Their bloodwas first lysed to remove RBCs, and then processed with iFCS deviceswithin 2 hours of blood draw. After iFCS enrichment, enriched cells werestained with epithelial marker (EpCAM), mesenchymal markers (vimentinand N-cadherin), leukocyte marker (CD45) as well as nucleus stainingDAPI for their identification. CTCs were identified as epithelialpositive (EpCAM+/CD45−/DAPI+), mesenchymal positive (Vim+/CD45−/DAPI+,N-cad+/CD45−/DAPI+ or Vim+/N-cad+/CD45−/DAPI+), or both epithelial andmesenchymal positive (EpCAM+/Vim+/N-cad+/CD45−/DAPI+), while WBCs wereidentified as CK−/Vim−/N-cad−/CD45+/DAPI+. Results from this study aresummarized in FIGS. 2.4A-2.4C. Examples of intact CTCs from deviceoutputs are shown in FIG. 2.4A and FIG. 3.5A. We first learned that theeffective diameter of CTCs, defined as maximum feret diameter of cellsfrom bright field images, showed a high degree of polydispersity amongthese enriched cells. For example, patient 1 of this cohort (BrC-P1-Opt)had an advanced stage breast cancer diagnosis (stage IIIA, pre-surgery).We identified 232 CTCs in 9.0 mL of blood (25 CTCs/mL) from thispatient. Effective diameters measured from randomly selected cells(n=24) of this patient were 11.99±7.87 μm (mean±s.d.), where thesmallest diameter was 4.95 μm and the largest was 33.11 μm (FIG. 2.4B).We characterized surface antigen expressions using above mentionedmarkers for 232 cells from this patient. The characterization revealed ahigh degree of heterogeneity of antigen expressions: 12.93% of them wasepithelial positive, 78.45% was mesenchymal positive, and 8.62% was bothepithelial and mesenchymal positive (FIG. 2.4C). Patient 2 of thiscohort (BrC-P2-Opt) had an early stage breast cancer diagnosis (stageIA, post-surgery). 82 CTCs were identified in 12.0 mL of blood (6CTCs/mL) from this patient. Effective diameters of CTCs (n=26) againshowed high polydispersity (mean±s.d.=13.73±6.76 μm, smallest diameter6.00 μm, and largest diameter 32.10 μm). Surface antigen expressions ofcells (n=82) revealed 54.88% was epithelial positive, 30.49% wasmesenchymal positive, and 14.63% was both epithelial and mesenchymalpositive. Similarly, a third post-surgery patient (BrC-P3-Opt) with astage IA breast cancer diagnosis exhibited variations in CTCs' size andheterogeneity among antigen expressions.

Overall, we found a variety of CTC subtypes that were positive foreither epithelial or mesenchymal factors alone, or cells that werepositive for both factors in this cohort (FIG. 2.4A). The cells thatwere positive for both factors likely represent CTCs that are intransition between Epithelial and Mesenchymal status, indicating theirevolution to more virulent tumor cell phenotypes. We found that eachpatient had a wide range in sizes of CTC's that overlapped with the sizedistribution of WBC's (FIG. 2.4B). This indicates that existingcell-size dependent methods could greatly decrease the sensitivity ofCTC enrichment, by excluding a large proportion of CTCs. Given theproportion of CTC subtypes in each patient, and the correspondingdistribution of cell sizes for each patient, a large proportion of thesize-excluded CTC's would have been mesenchymal (FIGS. 2.4B and 2.4C).We also observed that the relative numbers of CTC types varied greatlyamong patients (FIG. 2.4C). For example, “Patient 1” (BrC-P1-Opt) and“Patient 3” (BrC-P3-Opt) carried predominantly mesenchymal CTCs and“Patient 2” (BrC-P2-Opt) carried predominantly epithelial CTCs. In eachcase, the relative number of transitioning EMT cells (positive for bothepithelial and mesenchymal markers) was the least abundant withinpatient sample counts; however, the relative number of EMT cellssignificantly varied among patients. We went on to determine if thesevariable counts of CTC subtypes would correlate with clinical anddiagnostic variables in a third cohort at Henry Ford Health System(Detroit, Mich.). Explicitly, we postulated whether the patients withthe highest count of either mesenchymal or EMT cells would also have themost aggressive tumor phenotypes. These results will be discussed afternext section.

We extended our study to a second cohort including 3 non-surgical stageIV lung cancer patients. They were recruited and consented fromUniversity Cancer and Blood Center (Athens, Ga.) under the same IRBprotocol (University of Georgia, STUDY00005431). These patients areidentified as lung cancer optimization cohort (LC-P#-Opt, in which #indicates the number of patient). Same blood collection, processing andcell identification approaches were used as for breast cancer cohort,except cytokeratin (CK) replaced EpCAM as the epithelial marker. CTCswere identified as epithelial positive (CK+/CD45−/DAPI+), mesenchymalpositive (Vim+/CD45−/DAPI+, N-cad+/CD45−/DAPI+, orVim+/N-cad+/CD45−/DAPI+), or both epithelial and mesenchymal positive(CK+/Vim+/N-cad+/CD45−/DAPI−), while WBCs were identified asCK−/Vim−/N-cad−/CD45+/DAPI+. Results are summarized in FIGS. 2.5A-2.5Cwith intact CTCs being shown in FIG. 2.5A and FIG. 3.5B. We learned thatCTCs from lung cancer patients were highly variable in cell sizes andantigen expressions too. For example, patient 1 of this cohort(LC-P1-Opt) was diagnosed with advanced stage non-small cell lung cancer(stage IV). 228 CTCs were identified in 9.0 mL of blood (25 CTCs/mL).Effective diameters of CTCs (n=39) were 9.73±3.11 μm (mean±s.d.), wherethe smallest diameter was 4.59 μm and the largest was 18.52 μm. Surfaceantigen expression characterization of cells (n=228) showed that 11.84%was epithelial positive, 78.95% was mesenchymal positive, and 9.21% wasboth epithelial and mesenchymal positive. Data on the second patient(LC-P2-Opt, stage IV small cell lung cancer) and the third patient(LC-P3-Opt, stage IV non-small cell lung cancer) were consistent withthis observation. The study on both breast cancer and lung cancerpatients shows that CTCs from them are highly variable in cell diametersand in most cases their diameters overlap with contaminating WBCs (FIG.2.4B and FIG. 2.5B). Furthermore, surface antigen expressions of CTCsare non-uniform across cells, making methods relying solely on celldiameter or antigen expression ineffective. iFCS devices, insensitive toboth size and antigen variations, are able to enrich CTCs and preservethese variations.

Non-EpCAM Positive Type of CTCs Enriched by iFCS Show Better Correlationwith Pathological Variables in Early-Stage Breast Cancer Patients.

In the third cohort, we used iFCS devices at Henry Ford Health System(Detroit, Mich.) to process blood samples from 6 breast cancer patientswho were recruited and consented there under an approved IRB protocol(Henry Ford Health System, Davis-11564). These patients are identifiedas breast cancer culture cohort (BrC-P#-Culture, in which # indicatesthe number of patient). Peripheral blood was collected from the patientsbefore initiation of treatment. Similar blood collection, processing andcell identification approaches were used as in other cohorts. After iFCSenrichment, enriched cells were stained with epithelial marker (EpCAM),mesenchymal markers (vimentin), leukocyte marker (CD45) for theiridentification. CTCs were identified as epithelial positive(EpCAM+/CD45−), mesenchymal positive (Vim+/CD45−), or both epithelialand mesenchymal positive (EpCAM+/Vim+/CD45−), while WBCs were identifiedas CD45+. Results from this study are summarized in FIGS. 2.6A-2.6G.When we compared the number of each CTC subtype with clinical-pathologyvariables we found interesting correlations that suggest the non-EpCAMpositive type of CTCs, defined as vimentin-only positive and both EpCAMand vimentin positive, may have better prediction value with regard toprognosis and diagnosis, relative to epithelial (EpCAM-only positive)CTCs. Specifically, when we correlated the numbers of each CTC subtypewith tumor grade, we found that EpCAM-only CTCs were the leastcorrelated with this variable (R²=0.025) while mesenchymal cells(vimentin-only CTCs) were significantly more correlated (R²=0.584).Interestingly, the EMT cells had the highest correlation with grade(R²=0.734), suggesting that the presence of these transitioning cellsmay indicate the invasiveness and aggressiveness of the primary tumor(FIGS. 3.6A-3.6C).

We also investigated how relative CTC subtype counts correspond withstandard clinical diagnostic variables. We observed that of the patientswith the 21-gene recurrence risk scores (RS), the patient with highestscore (BrC-P6-Culture, RS=16) had the highest proportion of mesenchymalonly CTCs and also the lowest proportion of EMT CTCs. This may indicatethat the relative numbers of cells in transition vs cells that havecompletely transitioned to mesenchymal status, may be indicative ofmetastatic potential. Conversely, the patient with the lowest RS scorehad the highest proportion of epithelial CTCs and the largest proportionof EMT CTCs (FIG. 2.6C). Of all patients with RS values in this cohort,when we compared these with the relative numbers of CTC subtypes, wefound no significant correlation with this test. One limitation of thisnegative finding is that only 3 out of 6 patients had an indication forordering the 21-gene recurrence test and therefore we could notdetermine specific correlation of CTC subtypes with great confidence. Asan alternative to recurrence risk scores, we investigated whetherrelative correspondence of CTC subtypes with clinical stages had abetter correlation in these patients with recurrence risk estimates.Similar to our 21-gene recurrence test observations, we found that thepatient with the highest tumor stage (IIA) also had the highestproportion of mesenchymal-only CTCs and the lowest proportion ofepithelial CTCs (FIG. 2.6D). While the limitations of these comparisonspreclude statistical significance, there is a compelling trend ofspecific CTC subtypes correlated with clinical stage and prognosis amongthe small subset of patients.

Because iFCS allows us to obtain viable cells, we also cultured CTCs foreach patient in a pooled-subtype design, as opposed to subclones ofspecific subtypes. With these cultures, we investigated the relativepercentages of the CTC subtypes in each patient culture and measured thegrowth rate of the cultures over a 72-hour period. We had variablesuccess with culture growth (FIG. 2.6E) and this corresponded to avariable growth rates within cultures that grew significantly (FIG.2.6F). Of the patient samples that displayed significant growth curves(BrC-P3-Culture, BrC-P4-Culture, and BrC-P5-Culture) we found theirgrowth rate averaged over 60%. When we compared these growth rates withCTC subtype proportions, we found that the two patients with the highestgrowth curves had the lowest proportion of both epithelial and EMT CTCs,with the highest relative mesenchymal CTCs (of these we successfullyestablished cultures—patient BrC-P6-Culture had the highest mesenchymalproportion but the line was lost to contamination before a growth curvecould be calculated). In a preliminary comparison, we observed thatgrowth rates were positively correlated with the relative proportion ofmesenchymal cells (R²=0.289), though not significantly.

Discussion

We developed an iFCS method and its prototype devices for tumorantigen-independent and cell size variation-inclusive enrichment of CTCsfrom cancer patients. After validations with both spike-in samples andclinical samples, the performance of iFCS devices was determined to be:(1) a close-to-complete CTC recovery rate of 99.16% at clinicallyrelevant occurrence rate for CTCs (1-10 cells mL⁻¹), regardless of theirsurface antigens and sizes; (2) a minimal WBC contamination of 533±34cells at the device output for every one milliliter blood processed, (3)a blood processing throughput of 12 mL h⁻¹, and (4) minimally affectedcell integrity after enrichment, including viability and proliferation.We compared iFCS performance to a total of 36 recently reported CTCenrichment methods (Table 2.1) and found iFCS had better combinatorialperformance metrics in above-mentioned four categories than existingmethods. The only published CTC enrichment method with competingperformance was CTC-iChip.21-23 The operation of a most recent versionof CTC-iChip—monolithic CTC-iChip,21 integrated three working principlesincluding cell size based deterministic lateral displacement (DLD) todeplete red blood cells, inertial focusing to concentrate nucleatedcells, and magnetophoretic separation to enrich CTCs. The size based DLDstage risked depleting CTCs smaller than or of similar size as red bloodcells. In our iFCS study, we found that on average 34.5% of CTCs (33.1%for NSCLC CTCs, 36.4% for SCLC CTCs, and 34.6% for BrC CTCs) were lessthan 8 μm (Table 2.4). With other performance metrics (throughput, WBCcontamination, and cell integrity) being similar, iFCS could recover amore comprehensive repertoire of CTCs comparing to monolithic CTC-iChip.

We used iFCS devices to investigate whether heterogeneous populations ofCTC cell types could potentially yield clinical utility. From first twocohorts of cancer patients (breast and lung cancers), we discovered avariety of CTC subtypes that were positive for epithelial or mesenchymalbiomarkers alone, or cells that were positive for both markers. We alsodiscovered that each patient had a high level of CTC size variation,which overlapped with the size distribution of WBCs. This findinghighlights the need to develop tumor cell antigen independent and cellsize variation inclusive method for CTC studies.

Our third patient cohort—breast cancer cohort in this study are limitedto early-stage cancer patients that had no indication of metastaticdisease, which inherently allowed us to test whether iFCS devices wouldyield biomarkers that can be more informative about potential fordisease progression and/or undetected metastatic disease, includingmicrometastasis. The current clinical standard for utilization of CTCsis to rely on epithelial markers to select for tumor cells from theblood cell components. This EpCAM marker selection completely misses acritical component of the heterogenous CTC profile. By harvesting andcharacterizing the CTC subtype landscape from liquid biopsy, we candevelop a clinical application that assesses the ‘range’ and relativevariation in the numbers of mesenchymal cells, which we have currentlyshown may correlate with clinical prognostic indicators. For instance,with a single liquid blood biopsy, not only could we potentially detecttumor presence but also may be able to predict staging for the solidprimary tumor. This level of diagnostics is usually only accomplished byinvasive biopsy or surgery. In our assessment of recurrence scorecorrelations with CTC landscapes, certain patients were not given therecurrence risk testing because their staging was deemed too low.However, once we understand the variable landscapes associated with eachstage, recurrence estimates can be made more precisely from thesub-population of CTCs that would eventually evolve and establish distalmetastases. In this scenario, as Standard of Care disease management maydictate that low-staged patients are not candidates for recurrencetesting, a CTC profile could become a more appropriate indicator of theneed for recurrence testing, as opposed to detection of invasive tumormargins pre/post-surgical resection. In addition, while we see thatrecurrence scores appear not to be correlated to CTC landscape profiles,we consider that the genomic signatures that are used to derive theserecurrence risk scores are specific to the primary tumor. The source oftumor material is a core sample, which may not capture the heterogeneityof the most evolved or invasive region of the solid tumor. Therefore,the score may be different and more informative when assessing thesemetastatic-potential genes from CTCs and/or the leading edge of thetumor. Previous studies have shown that the spatial heterogeneity ofcells throughout the tumor are reflective of tumor evolution leading tothe most invasive regions and typically have a distinct expression andmutational profile, associated with tumor evolution and coupled diseaseprogression.24-26

The performance of iFCS opens up opportunities in precision medicinewhich address patient diversity. Specifically, accurately assessing thephenotypes of CTCs can be more reflective of metastatic potential andtherefore prognosis. In addition, understanding the mutational profileswithin CTCs can assist with creation of personalized cancer vaccinesthat exploit the distinct functional mutations that may occur inspecifically in CTCs (absent in the primary tumor) that subsequentlycolonize distal metastatic lesions.

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It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare set forth only for a clear understanding of the principles of thedisclosure. Many variations and modifications may be made to theabove-described embodiments of the disclosure without departingsubstantially from the spirit and principles of the disclosure. All suchmodifications and variations are intended to be included herein withinthe scope of this disclosure.

The invention claimed is:
 1. A cell separation system for enrichingcirculating tumor cells in a biological sample, the system comprising: aplurality of magnetic microbeads adapted for conjugation to white bloodcells in the biological sample, and not for conjugation with thecirculating tumor cells, to provide a magnetically labeled biologicalsample wherein a majority of the white blood cells in the biologicalsample are each conjugated to one or more of the plurality of magneticmicrobeads; a biocompatible superparamagnetic sheathing compositioncomprising a plurality of magnetic nanoparticles and a biocompatiblesurfactant, the biocompatible superparamagnetic sheathing compositionadapted to be combined with a biocompatible carrier fluid to make abiocompatible superparamagnetic sheathing fluid; and a multi-stagemicrofluidic device, the device comprising: (i) a filter sectioncomprising a first microfluidic channel, a first fluid inlet, and one ormore filters along a length of the first microfluidic channel, whereinthe first fluid inlet is configured to receive the magnetically labeledbiological sample, and wherein the one or more filters are configured toremove a first plurality of waste particles from the magneticallylabeled biological sample; (ii) a first separation stage comprising asecond microfluidic channel fluidly connected to the first microfluidicchannel and having a second fluid inlet to receive the biocompatiblesuperparamagnetic sheathing fluid, and a first fluid outlet at an end ofthe second microfluidic channel and offset from a central diameter ofthe second microfluidic channel, wherein the second microfluidic channelis configured to combine the magnetically labeled biological sample fromthe filter section with the biocompatible superparamagnetic sheathingfluid; (iii) a second separation stage comprising a third microfluidicchannel fluidly connected to the second microfluidic channel and havinga second fluid outlet and at least a first circulating tumor cell outletand second circulating tumor cell outlet; and (iv) one or more magneticsources adjacent to the multi-stage microfluidic device and configuredto produce: (a) a non-uniform magnetic field along the secondmicrofluidic channel having a component sufficiently perpendicular to alength of the second microfluidic channel to cause a plurality of whiteblood cells conjugated to the magnetic beads to be deflected into thefirst fluid outlet; and (b) a substantially symmetric magnetic fieldhaving a field maximum along a length of the third microfluidic channelsufficient to cause the white blood cells conjugated to the magneticbeads in the third microfluidic channel to be focused toward a center ofthe third microfluidic channel and to exit the channel via the secondfluid outlet and to cause circulating tumor cells to be deflectedtowards an outer portion of the third microfluidic channel and to exitthe channel via the first or second circulating tumor cell outlet. 2.The cell separation system of claim 1, wherein the multi-stagemicrofluidic device has a serpentine shape and wherein: the filtersection comprises a first end and a second end fluidly connected by thefirst microfluidic channel, wherein the first fluid inlet is fluidlyconnected to the first microfluidic channel at the first end, and theone or more filters are located between the first and second end of thefirst microfluidic channel; the first separation stage comprises a thirdend and fourth end fluidly connected by the second microfluidic channel,wherein the third end is connected to the second end of the firstmicrofluidic channel of the filter section by a first u-shaped channel,wherein the second fluid inlet is fluidly connected to the secondmicrofluidic channel at the third end and the first fluid outlet isfluidly connected to the second microfluidic channel at the fourth end;and the second separation stage comprises a fifth end and a sixth endfluidly connected by the third microfluidic channel, wherein the fifthend is connected to the fourth end of the second microfluidic channel ofthe first separation stage by a second u-shaped channel, wherein thesecond fluid outlet is fluidly connected to the third microfluidicchannel at the sixth end and configured to receive materials flowingthrough a central portion of the third microfluidic channel, and whereinthe first and second circulating tumor cell outlets are each fluidlyconnected to the third microfluidic channel at the sixth end and offsetfrom the center of the third microfluidic channel and configured toreceive material flowing near the outer portion of the thirdmicrofluidic channel.
 3. The cell separation system according to claim1, wherein the first fluid outlet is offset from a central diameter ofthe second microfluidic channel and configured to receive white bloodcells conjugated to magnetic beads and flowing on one side of the secondmicrofluidic channel.
 4. The cell separation system according to claim1, wherein the one or more magnetic sources comprises a first magnetarray and a second magnet array arranged in a quadrupole configurationsuch that the first magnet array and the second magnet array areoriented to repel each other; wherein the second separation stage isbetween the first magnet array and the second magnet array and orientedsuch that the length of the third microfluidic channel is centrallyaligned between the first magnet array and the second magnet array. 5.The cell separation system according to claim 4, wherein a verticaldistance between the third microfluidic channel and each of the firstmagnet array and the second magnet array is about 800-1200 μm.
 6. Thecell separation system according to claim 1, wherein one or more of thefirst microfluidic channel, the second microfluidic channel, and thethird microfluidic channel have a thickness of about 100 μm to about 500μm.
 7. The cell separation system according to claim 1, wherein thesecond microfluidic channel has a width of about 1400 μm to 1800 μm andthe third microfluidic channel has a width of about 1000 μm to 1400 μm.8. The cell separation system according to claim 1, wherein thecirculating tumor cells that exit via the first and second circulatingtumor cell outlet comprise about 97%, or more, of the total number ofcirculating tumor cells present in the biological sample inserted intothe first fluid inlet when in operation.
 9. The cell separation systemaccording to claim 1, wherein the biological sample is selected from thegroup consisting of: whole blood and whole blood that has been treatedwith lysis buffer to lyse red blood cells, wherein the biological samplecomprises a plurality of components.
 10. The cell separation systemaccording to claim 1, wherein about 95% to 100%, of the white bloodcells are each conjugated to one or more of the plurality of magneticmicrobeads.
 11. The cell separation system according to claim 1, whereina majority of the white blood cells in the magnetically labeledbiological sample are each conjugated to about 1 to about 60 magneticmicrobeads.
 12. The cell separation system according to claim 1, whereina majority of the white blood cells in the magnetically labeledbiological sample are each conjugated to an average of about 20-50magnetic microbeads.
 13. The cell separation system according to claim1, wherein the one or more magnetic sources produce a flux density ofabout 0.4 to 0.6T in the first separation stage and about 1.3 to 1.5 Tin the second separation stage.
 14. The cell separation system accordingto claim 1, wherein the one or more magnetic sources produce a fluxdensity gradient that ranges from about 4 to 256 T m⁻¹ in the firstseparation stage and from about 0-625 T m⁻¹ in the second separationstage, wherein the flux density gradient is lower at the center of thethird microfluidic channel of the second separation stage than at theouter portion of the third microfluidic channel of the second separationstage.
 15. The cell separation system according to claim 1, wherein thebiological sample comprises about 1 to 1000 circulating tumor cells permilliliter of the biological sample.
 16. The cell separation systemaccording to claim 1, wherein the circulating tumor cells are selectedfrom the group consisting of a primary cancer cell, a lung cancer cell,a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell,and a combination thereof.
 17. The cell separation system according toclaim 1, wherein the plurality of magnetic microbeads are conjugatedwith leukocyte antibodies for binding white blood cells in thebiological sample.
 18. A multi-stage microfluidic device for enrichingcirculating tumor cells in a biological sample, the device comprising:(i) a filter section comprising a first microfluidic channel, a firstfluid inlet, and one or more filters along a length of the firstmicrofluidic channel, wherein the first fluid inlet is configured toreceive a biological sample comprising circulating tumor cells and whiteblood cells, wherein a majority of the white blood cells in thebiological sample are each conjugated to magnetic microbeads, andwherein the one or more filters are configured to remove a firstplurality of waste particles from the biological sample; (ii) a firstseparation stage comprising a second microfluidic channel fluidlyconnected to the first microfluidic channel and having a second fluidinlet to receive a biocompatible superparamagnetic sheathing fluid, anda first fluid outlet at an end of the second microfluidic channelopposite the second fluid inlet and offset from a central diameter ofthe second microfluidic channel, wherein the second microfluidic channelis configured to combine the biological sample from the first filtersection with the biocompatible superparamagnetic sheathing fluid, andwherein the biocompatible superparamagnetic sheathing fluid comprises aplurality of magnetic nanoparticles and a biocompatible surfactantsuspended in a biocompatible carrier fluid; (iii) a second separationstage comprising a third microfluidic channel fluidly connected to thesecond microfluidic channel and having a second fluid outlet and atleast a first circulating tumor cell outlet and second circulating tumorcell outlet; and (iv) one or more magnetic sources adjacent to themulti-stage microfluidic device and configured to produce: (a) anon-uniform magnetic field along the second microfluidic channel havinga component sufficiently perpendicular to a length of the secondmicrofluidic channel to cause a plurality of white blood cellsconjugated to the magnetic beads to be deflected into the first fluidoutlet; and (b) a substantially symmetric magnetic field having a fieldmaximum along a length of the third microfluidic channel sufficient tocause the white blood cells conjugated to the magnetic beads in thethird microfluidic channel to be focused toward a center of the thirdmicrofluidic channel and to exit the channel via the second fluid outletand to cause circulating tumor cells to be deflected towards an outerportion of the third microfluidic channel and to exit the channel viathe first or second circulating tumor cell outlet.
 19. The multi-stagemicrofluidic device of claim 18, wherein the device has a serpentineshape and wherein the filter section is fluidly connected to the firstseparation stage by a first u-shaped channel and wherein the firstseparation stage is fluidly connected to the second separation stage bya second u-shaped channel.
 20. The multi-stage microfluidic device ofclaim 18, wherein the one or more magnetic sources comprise a firstmagnet array and a second magnet array arranged in a quadrupoleconfiguration such that the first magnet array and the second magnetarray are oriented to repel each other; wherein the second separationstage is between the first magnet array and the second magnet array andoriented such that the length of the third microfluidic channel iscentrally aligned between the first magnet array and the second magnetarray.